Carboxy x rhodamine analogs

ABSTRACT

The present invention provides novel fluorescent dyes and kits containing the same, which are useful for labeling a wide variety of biomolecules, cells and microorganisms. The present invention also provides various methods of using the fluorescent dyes for research and development, forensic identification, environmental studies, diagnosis, prognosis and/or treatment of disease conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/682,589, filed Nov. 20, 2012, which claims priority to U.S.Provisional Application No. 61/562,021, filed Nov. 21, 2011, the entirecontents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to fluorescent dyes and methods of using them.

BACKGROUND

Fluorescent dyes are widely used in biological research and medicaldiagnostics. Fluorescent dyes tend to be superior to conventionaltechniques because they are less expensive, less toxic and can generallybe detected with sufficient sensitivity. A diversity of fluorescent dyeswith a distinguishable color range has made it more practical to performmultiplexed assays capable of detecting multiple biologic targets at thesame time.

Further improvement in the properties of the dyes is needed in order tomeet the increasing demands of new instruments and new biologicalapplications. In particular, additional strategies to allow forfine-tuning of the wave-lengths of the dyes for maximal signal detectionand to provide additional colors are needed.

SUMMARY

In one aspect, the invention provides a compound according to formula(Ia) or (Ib):

where

R¹ and R¹¹ are independently H or C₁₋₄ alkyl, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R², R⁵, R¹² and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H,SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, and R²⁶ are independently H or C₁₋₆ alkylor one or more of R²⁰ and R²¹, R²¹ and R²², R²² and R²³, R²⁴ and R²⁵,R²⁵ and R²⁶, and R²⁶ and R²³ together form an aryl, heteroaryl,carbocyclic or heterocyclic ring;

R¹ and R² and/or R¹¹ and R¹² may together form a carbocyclic,heterocyclic, aryl or heteroaryl ring;

R⁶⁻¹⁰ are independently H, halo, OH, alkyl, aryl, heteroaryl, CO₂H,SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

each X is independently CHR²³, O, S or NR³⁰; and

R³⁰ is H, C₁₋₄ alkyl or —C(O)C₁₋₄ alkyl.

In another aspect, the invention provides a compound according toformula (IIa) or (IIb):

wherein

R¹ and R¹¹ are independently H or C₁₋₄ alkyl, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R², R³, R₄, R₅, R₁₂, R₁₃, R₁₄ and R₁₅ are independently H, alkyl, aryl,heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

R²⁰, R²¹, R²², R²³, R²⁴, R²⁵ and R²⁶ are independently H or C₁₋₆ alkylor one or more of R²⁰ and R²¹, R²¹ and R²², R²² and R²³, R²⁴ and R²⁵,R²⁵ and R²⁶, and R²⁶ and R²³ together form an aryl, heteroaryl,carbocyclic or heterocyclic ring;

R¹ and R² and/or R¹¹ and R¹² may together form a carbocyclic,heterocyclic, aryl or heteroaryl ring;

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

each X is independently CHR²³, O, S or NR³⁰; and

R³⁰ is H, C₁₋₄ alkyl or —C(O)C₁₋₄ alkyl.

In a further aspect, the invention provides a compound according toformula (IIIa), (IIIb) or (IIIc):

wherein

R¹¹ is independently H or C₁₋₄ alkyl, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R² and R¹⁶ can be independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

R³ and R⁴ are H, alkyl, L-R, L-C_(s), L-CO₂H, L-SO₃H or together form acarbocyclic, aryl, heteroaryl, or heterocyclic ring;

alternatively, R² and R³ and independently R⁴ and R¹⁶ together form acarbocyclic, heterocyclic, aryl or heteroaryl ring;

R⁵, R¹², R¹³, R¹⁴ and R¹⁵ are independently H, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

R²⁰, R²¹, R²² and R²³ are independently H or C₁₋₆ alkyl or one or moreof R²⁰ and R²¹, R²¹ and R²², R²² and R²³, together form an aryl,heteroaryl, carbocyclic or heterocyclic ring;

R¹¹ and R¹² may together form a carbocyclic, heterocyclic, aryl orheteroaryl ring;

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

X is CHR²³, O, S or NR³⁰; and

R³⁰ is H, C₁₋₄ alkyl or —C(O)C₁₋₄ alkyl.

In yet another aspect, the invention provides a compound according toformula (IV):

wherein

R² and R¹⁶ can be independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R³ and R⁴ are H, alkyl or together form a carbocyclic, aryl, heteroaryl,or heterocyclic ring;

alternatively, R² and R³ and independently R⁴ and R¹⁶ together form acarbocyclic, heterocyclic, aryl or heteroaryl ring;

R⁵ and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

R²² and R²³ are independently H or C₁₋₆ alkyl or together form an aryl,heteroaryl, carbocyclic or heterocyclic ring; and

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s).

In a further aspect, the invention provides a compound according toformula (V):

wherein

R² and R¹⁶ can be independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R³ and R⁴ are H, alkyl, or together form a carbocyclic, heterocyclic ,aryl or heteroaryl ring; alternatively, R² and R³ and independently R⁴and R¹⁶ may together form a carbocyclic, heterocylic, aryl or heteroarylring;

R⁵ and R¹⁵ are independently H, alkyl, aryl, heteoaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

R²⁰ and R²¹ are independently H or C₁₋₆ alkyl or together form an aryl,heteroaryl, carbocyclic or heterocyclic ring; and

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s).

In another aspect, the invention provides a compound according toformula (VI):

wherein

R⁵ and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R²², R²³, R²⁶ and R²⁷ are independently H or C₁₋₆ alkyl or one or moreof R²² and R²³ and R²⁶ and R²⁷ together form an aryl, heteroaryl,carbocyclic or heterocyclic ring;and

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s).

In an additional aspect, the invention provides a compound according toformula (VII):

wherein

R⁵ and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R²⁰, R²¹, R²⁴ and R²⁵ are independently H or C₁₋₆ alkyl or one or moreof R²⁰ and R²¹ and R²⁴ and R²⁵ together form an aryl, heteroaryl,carbocyclic or heterocyclic ring; and

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s).

In a further aspect, the invention provides a compound of formula(VIIIa) or (VIIIb):

wherein

R¹¹ is H or C₁₋₄ alkyl, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R¹² and R¹⁵ are independently H, alkyl, aryl, CO₂H, SO₃H, L-CO₂H,L-SO₃H, L-R or L-C_(s);

R²⁰, R²¹, R²² and R²³ are independently H or C₁₋₆ alkyl or one or moreof R²⁰ and R²¹,

R²¹ and R²² and R²² and R²³ together form a fused aryl ring;

R¹¹ and R¹² may be joined together in an optionally substituted ring;

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

X is CHR²³, O, S or NR³⁰; and

R³⁰ is H, C₁₋₄ alkyl or —C(O)C₁₋₄ alkyl.

In some aspects, the invention provides a labeled biomolecule. In someaspects, the labeled biomolecule is a small molecule, e.g., a drug ordrug compound. In other aspects, the labeled biomolecule or labeledsmall molecule acts as a fluorescent tracer to monitor binding to atarget, e.g., drug target.

In other aspects, the invention provides a method to detect a selectedmolecule in a sample, comprising: a) contacting a sample suspected ofcontaining a selected molecule with a composition comprising a conjugatecomprising a compound according to the present invention and a ligandfor the selected molecule so as to yield a mixture; and b) detecting thepresence or amount of the compound in the mixture.

In some aspects, the invention provides a method of detecting thepresence of a nucleic acid polymer in a sample comprising: contacting asample suspected of containing a nucleic acid polymer with a compositioncomprising a conjugate comprising a compound according to the presentinvention and an oligonucleotide; and detecting the presence or amountof the compound in the sample.

In some aspects, the invention provides a method of monitoring bindingto a target of interest, e.g., a drug target, comprising contacting asample comprising a fusion protein comprising the target of interestwith a small molecule conjugated to a dye described herein; anddetecting binding of the small molecule conjugate to the target ofinterest. In some aspects, the fusion protein comprises a luciferaseprotein fused to the target of interest. In other aspects, the fusionprotein comprises a fluorescent protein fused to the target of interest.In some aspects, wherein the fusion protein comprises a luciferaseprotein, binding is detected by bioluminescence resonance energytransfer (BRET). In some aspects, wherein the fusion protein comprises afluorescent protein, binding is detected by fluorescent resonance energytransfer (FRET). In some aspects, the sample comprises a cell expressingthe fusion protein.

In some aspects, the invention provides a method for monitoringprotein-protein interactions comprising contacting a sample comprising afirst fusion protein and a second fusion protein with a ligandconjugated to a dye described herein; and detecting the interactionbetween the first and second fusion protein. In some aspects, the firstfusion protein comprises a luciferase protein fused to a first bindingpartner, the second fusion protein comprises a HaloTag® protein fused toa second binding partner, and the ligand conjugate comprises a HaloTag®ligand.

In some aspects, the invention provides reactive dyes that may be usedto label a protein(s), peptide(s) or ligand(s). In some aspects, thereactive dyes of the invention could be attached to a target protein orpeptide using a reactive cyanobenzothiazole labeling chemistry.

In other aspects, the invention provides a kit comprising a compoundaccording to the present invention or a labeled biomolecule according tothe present invention. In some aspects, the kit comprises a labeledbiomolecule or labeled small molecule, e.g., drug or drug compound. Insome aspects, the kit further comprises cells expressing a fusionprotein comprising a protein or target of interest. In other aspects,the kit further comprises a vector for expressing a fusion proteincomprising a protein or target of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E shows various compounds according to the present invention.

FIGS. 2A-2E shows various compounds according to the present invention.

FIGS. 3A-3E shows various compounds according to the present invention.

FIG. 4 shows electropherograms showing peaks of loci labeled with FAM,JOE, ET TMR, ET ROX and ET 4510. Male DNA (1.0 ng) was amplified using a21-STR Primer Pair Mix (Table 1) and the additional loci D22S1045,D2S441 and DYS391. Amplification products were analyzed with an AppliedBiosystems 3500xL Genetic Analyzer. Panel A. An electropherogram showingthe peaks of the FAM-labeled loci: Amelogenin, D3S1358, D1S1656,D6S1043, D13S317 and Penta E. Panel B. An electropherogram showing thepeaks of the JOE-labeled loci: Penta D, D16S539, D18S51, D2S1338 andCSF1PO. Panel C. An electropherogram showing the peaks of the ETTMR-labeled loci: THO1, vWA, D21S11, D7S820, D5S818, and TPDX. Panel D.An electropherogram showing ET ROX-labeled loci: D8S1179, D12S391,D19S433 and FGA. Panel E. An electropherogram showing ET 4510-labeledloci: D22S1045, D2S441 and DYS391. Note: Penta D is the largest loci inthe JOE-labeled loci set, and the ET 4510-labeled loci are shown fromsmallest to largest. Loci in the other sets are shown from largest tosmallest.

FIG. 5 shows electropherograms showing peaks of the loci DYS391, D2S441and D22S1045 labeled with 4510 (Panel A), 4563 (Panel B) or 4574 (PanelC). Male DNA (1.0 ng) was amplified using a 21-STR Primer Pair Mix(Table 1) and the additional loci D22S1045, D2S441 and DYS391.Amplification products were analyzed with an Applied Biosystems 3500 xLGenetic Analyzer.

FIG. 6 shows electropherograms showing peaks of the loci DYS391, D2S441and D22S1045 labeled with 4510 (Panel A), 4563 (Panel B), or 4574 (PanelC) and the JOE labeled loci: Penta D, D16S539, D18S51, D2S1338 andCSF1PO. Male DNA (1.0 ng) was amplified using a 21-STR Primer Pair Mix(Table 1) and the additional loci D22S1045, D2S441 and DYS391.Amplification products were analyzed with an Applied Biosystems 3500 xLGenetic Analyzer.

FIG. 7 shows electropherograms showing peaks of the loci DYS391, D2S441and D22S1045 labeled with 4510 (Panel A), 4563 (Panel B), or 4574 (PanelC). Male DNA (0.5 ng) was amplified using a 21-STR Primer Pair Mix(Table 1) and the additional loci D22S1045, D2S441 and DYS391.Amplification products were analyzed with an Applied Biosystems 3500 xLGenetic Analyzer.

FIG. 8 shows electropherograms showing peaks of the loci DYS391, D2S441and D22S1045 labeled with 4510 (Panel A), 4563 (Panel B), or 4574 (PanelC) and the JOE labeled loci: Penta D, D16S539, D18S51, D2S1338 andCSF1PO. Male DNA (0.5 ng) was amplified using a 21-STR Primer Pair Mix(Table 1) and the additional loci D22S1045, D2S441 and DYS391.Amplification products were analyzed with an Applied Biosystems 3500 xLGenetic Analyzer.

FIGS. 9( a)-9(d) shows confocal images using ligand 3780. FIG. 9( a)U2OS cells stably expressing HaloTag® containing a nuclear localizationsequence (HT-NLS) were labeled with 100 nM ligand 3780 by a no-washprotocol and imaged using 3% λ633 laser, PMT 715, CA 80 μm, 100×. FIG.9( b) U2OS cells stably expressing HT-NLS were labeled with 1 μM ligand3780 by a rapid label protocol and imaged using 3% λ633 laser, PMT 600,CA 200 μm, 100×. FIG. 9( c) U2OS cells stably expressing the fusionprotein p65-HaloTag (p65-HT) were labeled with 1 μM ligand 3780 by arapid label protocol and imaged using 8% λ633 laser, PMT 775, CA 80 μm,100×. FIG. 9( d) U2OS cells labeled with 1 μM ligand 3780 by a rapidlabel protocol and imaged using 8% λ633 laser, PMT 775, CA 200 μm, 20×.The left panel in each image shows fluorescence channel, the middlepanel shows DIC, and the right panel shows an overlay of the two.

FIGS. 10( a)-10(b) shows confocal images using ligand 3781. FIG. 10( a)U2OS cells stably expressing the fusion protein p65-HT and FIG. 10( b)U2OS cells were labeled with 1 μM ligand 3781 by a rapid label protocol.Cells were imaged using 10% λ543 laser, PMT 830, CA 80 μm, 80×. The leftpanel in each image shows fluorescence channel, the middle panel showsDIC, and the right panel shows an overlay of the two.

FIGS. 11( a)-11(d) shows confocal images using ligand 3782. FIG. 11( a)U2OS cells stably expressing HT-NLS were labeled with 100 nM ligand 3782by a no-wash protocol and imaged using 3% λ633 laser, PMT 615, CA 80 μm,100×. FIG. 11( b) U2OS cells stably expressing HT-NLS were labeled with1 μM ligand 3782 by a rapid label protocol and imaged using 3% λ633laser, PMT 600, CA 200 μm, 100×. FIG. 11( c) U2OS stably expressing thefusion protein p65-HT were labeled with 1 μM ligand 3782 by a rapidlabel protocol and imaged using 8% λ633 laser, PMT 775, CA 80 μm, 100×.FIG. 11( d) U2OS cells were labeled with 1 μM ligand 3782 by a rapidlabel protocol and imaged using 8% λ633 laser, PMT 775, CA 200 μm, 20×.The left panel in each image shows fluorescence channel, the middlepanel shows DIC, and the right panel shows an overlay of the two.

FIGS. 12( a)-12(d) shows confocal images using ligand 3783. FIG. 12( a)U2OS cells stably expressing HT-NLS were labeled with 100 nM ligand 3783by a no-wash protocol and imaged using 3% λ633 laser, PMT 615, CA 80 μm,100×. FIG. 12( b) U2OS cells stably expressing HT-NLS were labeled with1 μM ligand 3783 by a rapid label protocol and imaged using 3% λ633laser, PMT 600, CA 200 μm, 100×. FIG. 12( c) U2OS cells stablyexpressing the fusion protein p65-HT were labeled with 1 μM ligand 3783by the rapid label protocol and imaged using 8% λ633 laser, PMT 750, CA80 μm, 100×. FIG. 12( d) U2OS cells were labeled with 1 μM ligand 3783by a rapid labeled protocol and imaged using 8% λ633 laser, PMT 775, CA200 μm, 20×. The left panel in each image shows fluorescence channel,the middle panel shows DIC, and the right panel shows an overlay of thetwo.

FIGS. 13( a)-13(c) shows confocal images using ligand 3905. FIG. 13( a)U2OS cells stably expressing HT-NLS were labeled with 1 μM ligand 3905by a rapid label protocol and imaged using 3% λ633 laser, PMT 600, CA200 μm, 20×. FIG. 13( b) U2OS cells stably expressing the fusion proteinp65-HT and FIG. 13( c) U2OS cells were labeled with 1 μM ligand 3905 bya rapid label protocol and imaged using 15% λ633 laser, PMT800, CA 80μm, 20×. The left panel in each image shows fluorescence channel, themiddle panel shows DIC, and the right panel shows an overlay of the two.

FIGS. 14( a)-14(c) shows confocal images using ligand 3906. FIG. 14( a)U2OS cells stably expressing HT-NLS were labeled with 1 μM ligand 3906by a rapid label protocol and imaged using 3% λ633 laser, PMT 600, CA200 μm, 20×. FIG. 14( b) U2OS cells stably expressing the fusion proteinp65-HT and FIG. 14( c) U2OS cells were labeled with 1 μM ligand 3906 bya rapid label protocol and imaged using 10% λ633 laser, PMT 720, CA 80μm, 20×. The left panel in each image shows fluorescence channel, themiddle panel shows DIC, and the right panel shows an overlay of the two.

FIGS. 15( a)-15(b) shows confocal images of ligand 3954. FIG. 15( a)U2OS cells stably expressing HT-NLS and FIG. 15( b) U2OS cells werelabeled with 1 μM ligand 3954 by a rapid label protocol and imaged using4% λ633 laser, PMT 880, CA 80 μm, 20×. The left panel in each imageshows fluorescence channel, the middle panel shows DIC, and the rightpanel shows an overlay of the two.

FIGS. 16( a)-16(b) shows confocal images of ligand 4356 and 4357. U2OScells stably expressing HT-NLS were labeled with 1 μM of ligand FIG. 16(a) 4356 or FIG. 16( b) 4357 by a rapid label protocol and imaged using10% λ633 laser, PMT 830, CA 80 μm, 30×. The left panel in each imagesshows fluorescence channel, the middle panel shows DIC, and the rightpanel shows an overlay of the two.

FIGS. 17( a)-17(b) shows the labeling efficiency of ligands 3780, 3782and 3783 in U2OS cells stably expressing HT-NLS. FIG. 17( a) Fluorescentscans after SDS-PAGE showing ligand signal directly (λ633) and TMRsignal (λ532). Lane 1 represents 100 nM ligand using a no-wash labeling(pulse) with 5 μM TMR rapid labeling (chase), lane 2 represents 1 μMrapid labeling (pulse) with 5 μM TMR rapid labeling (chase), lane 3represents 5 μM rapid labeling (pulse) with 5 μM TMR rapid labeling(chase), “TMR” represents cells labeled only with TMR ligand. FIG. 17(b) Graph showing quantification of bands (TMR signal) from TMR gel as apercent of TMR alone band.

FIG. 18 shows viability of U2OS and CHO cells in the presence of ligand3782. Graph shows results of CellTiter-Glo® Luminescent Cell ViabilityAssay after 24 hour incubation with ligand 3782 or DMSO carrier(control). Each bar represents n=6 wells, ±SEM.

FIG. 19 shows the performance of ligand 3782 in a gel-based analysis asan alternative to the HaloTag® TMR ligand.

FIG. 20 shows a schematic representation of a p38alpha kinasefluorescent tracer PBI 4838 binding to an inactive p38alpha kinase.

FIGS. 21A-21B shows a titration of the dye tracer PBI 4838 with cellsexpressing a NanoLuc-p38 fusion protein monitored using BRET. Inaddition, the figure shows that the interaction of the tracer andNanoLuc-p38 fusion can be inhibited by BIRB796. Binding was monitoredusing BRET in lysed cells FIG. 21A and live cells FIG. 21B.

FIGS. 22A-22B shows monitoring of the binding of a known drug, BIRB796,to a kinase target, p38, in living cells using a fluorescent dye tracerPBI 4838. Binding was monitored using BRET and the EC50 value determinedfor p38 FIG. 21A and PKCa (negative control) FIG. 21B.

FIGS. 23A-23B shows a rapamycin-dependent increase of BRET occurringbetween a NanoLuc-Frb fusion (donor) and a HaloTag-FKBP12 fusion boundto PBI 3781, a dye conjugated HaloTag® ligand FIG. 23A. FIG. 23B showsthe emission spectra for NanoLuc luciferase and PBI 3781 and excitationspectra for PBI 3781

FIG. 24 shows the monitoring of the rapamycin-mediated interactionbetween a NanoLuc-Frb fusion and HaloTag-FKPf12 fusion using BRET and adye conjugated HaloTag® ligand (PBI 3781).

DETAILED DESCRIPTION Definitions

As used herein, the following terms and expressions have the indicatedmeanings. Specific values listed below for radicals, substituents, andranges are for illustration only. They do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents.

When a group or moiety can be substituted, the term “substituted”indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in someembodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens onthe group indicated in the expression using “substituted” can bereplaced with a selection of recited indicated groups or with a suitablegroup known to those of skill in the art (e.g., one or more of thegroups recited below), provided that the indicated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.Suitable substituents of a substituted group can include alkyl, alkenyl,alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl,heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino,alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl,acetylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy,carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl,arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl,heterocyclesulfinyl, heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl (alkyl)amine, and cyano. Additionally, the suitableindicated groups can include, e.g., —X, —R, —O—, —OR, —SR, —S—, —NR₂,—NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃,NC(═O)R, —C(═O)R, —C(═O)NRR —S(═O)₂O—, —S(═O)₂OH, —S(═O)₂R, —OS(═O)₂OR,—S(═O)₂NR, —S(═O)R, —OP(═O)O₂RR, —P(═O)O₂RR, —P(═O)(O—)₂, —P(═O)(OH)₂,—C(═O)R, —C(═O)X, —C(S)R, —C(O)OR, —C(O)O—, —C(S)OR, —C(O)SR, —C(S)SR,—C(O)NRR, —C(S)NRR, —C(NR)NRR, where each X is independently a halogen(“halo”): F, Cl, Br, or I; and each R is independently H, alkyl, aryl,heteroaryl, heterocycle, a protecting group or prodrug moiety. As wouldbe readily understood by one skilled in the art, when a substituent isketo (═O) or thioxo (═S), or the like, then two hydrogen atoms on thesubstituted atom are replaced.

As used herein, the term “alkyl” refers to a branched, unbranched, orcyclic hydrocarbon having, for example, from 1 to 20 carbon atoms, andoften 1 to 12, or 1 to about 6 carbon atoms. Examples include, but arenot limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl,2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl (t-butyl), 1-pentyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl,2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl,3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. Thealkyl can be unsubstituted or substituted. For example, a substitutedalkyl group can be a haloalkyl group, as described below. The alkyl canalso be optionally partially or fully unsaturated. As such, therecitation of an alkyl group includes both alkenyl and alkynyl groups.The alkyl can be a monovalent hydrocarbon radical, as described andexemplified above, or it can be a divalent hydrocarbon radical (i.e.,alkylene), according to the context of its usage. Additionally, thealkyl group can be optionally interrupted, as described below for theterm interrupted.

The term “alkenyl” refers to a monoradical branched or unbranchedpartially unsaturated hydrocarbon chain (i.e. a carbon-carbon, sp²double bond). In one embodiment, an alkenyl group can have from 2 to 10carbon atoms, or 2 to 6 carbon atoms. In another embodiment, the alkenylgroup has from 2 to 4 carbon atoms. Examples include, but are notlimited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, and thelike. The alkenyl can be unsubstituted or substituted.

The term “alkynyl” refers to a monoradical branched or unbranchedhydrocarbon chain, having a point of complete unsaturation (i.e. acarbon-carbon, sp triple bond). In one embodiment, the alkynyl group canhave from 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In anotherembodiment, the alkynyl group can have from 2 to 4 carbon atoms. Thisterm is exemplified by groups such as ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,1-octynyl, and the like. The alkynyl can be unsubstituted orsubstituted.

The term “cycloalkyl” or “carbocyle” or “carbocyclic” refers to cyclicalkyl groups of from 3 to about 10 carbon atoms having a single cyclicring or multiple condensed rings. Such cycloalkyl groups include, by wayof example, single ring structures such as cyclopropyl, cyclobutyl,cyclopentyl, cyclooctyl, and the like, or multiple ring structures suchas adamantanyl, and the like. The cycloalkyl can be unsubstituted orsubstituted. The cycloalkyl group can be monovalent or divalent, and canbe optionally substituted as described above for alkyl groups. Thecycloalkyl group can optionally include one or more cites ofunsaturation, for example, the cycloalkyl group can include one or morecarbon-carbon double bonds, such as, for example, cyclohexene,1,3-cyclohexadiene, 1,4-cyclohexadiene, and the like. The cycloalkylgroup can be a carbocycle, which refers to a saturated or partiallyunsaturated ring having 3 to 8 carbon atoms as a monocycle, 7 to 12carbon atoms as a bicycle, and up to about 20 carbon atoms as apolycycle. Monocyclic carbocycles typically have 3 to 6 ring atoms,still more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to12 ring atoms, e.g., arranged as a bicyclo[4,5], [5,5], [5,6] or [6,6]system, or 9 or 10 ring atoms arranged as a bicyclo[5,6] or [6,6]system. Examples of carbocycles include cyclopropyl, cyclobutyl,cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl,cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, or 1-cyclohex-3-enyl.The carbocycle can be optionally substituted as described above foralkyl groups.

The term “alkoxy” refers to the group alkyl-O-, where alkyl is asdefined herein. In one embodiment, alkoxy groups include, e.g., methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexyloxy, 1,2-dimethylbutoxy, and the like. The alkoxy canbe unsubstituted or substituted.

The term “aryl” refers to an aromatic hydrocarbon group derived from theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. The radical can be at a saturated or unsaturatedcarbon atom of the parent ring system. The aryl group can have 6-18carbon atoms, 6-14 carbon atoms, or 6-10 carbon atoms. The aryl groupcan have a single ring (e.g., phenyl) or multiple condensed (fused)rings, wherein at least one ring is aromatic (e.g., naphthyl,dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groupsinclude, but are not limited to, radicals derived from benzene,naphthalene, anthracene, biphenyl, and the like. The aryl can beunsubstituted or optionally substituted, as described above for alkylgroups. For example, an aryl group can be substituted with one or moresubstituents (as described above) to provide various substituted aryls,such as pentafluorophenyl or para-trifluoromethylphenyl, and the like.

The term “halo” refers to the groups fluoro, chloro, bromo, and iodo.Similarly, the term “halogen” refers to fluorine, chlorine, bromine, andiodine.

The term “heteroaryl” is defined herein as a monocyclic, bicyclic, ortricyclic ring system containing one, two, or three aromatic rings andcontaining at least one nitrogen, oxygen, or sulfur atom in an aromaticring, and that can be unsubstituted or substituted, for example, withone or more, and in particular one to three, substituents, as describedabove in the definition of “substituted”. Typical heteroaryl groupscontain 2-14 carbon atoms in addition to the one or more heteroatoms.Examples of heteroaryl groups include, but are not limited to,2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl,benzothiazolyl, β-carbolinyl, carbazolyl, chromenyl, cinnolinyl,dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl,indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl,pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl,tetrazolyl, and xanthenyl. In one embodiment the term “heteroaryl”denotes a monocyclic aromatic ring containing five or six ring atomscontaining carbon and 1, 2, 3, or 4 heteroatoms independently selectedfrom non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H,O, alkyl, aryl, or (C₁-C₆)alkylaryl. In another embodiment heteroaryldenotes an ortho-fused bicyclic heterocycle of about eight to ten ringatoms derived therefrom, particularly a benz-derivative or one derivedby fusing a propylene, trimethylene, or tetramethylene diradicalthereto.

The term “heterocycle” refers to a saturated or partially unsaturatedring system, containing at least one heteroatom selected from the groupoxygen, nitrogen, and sulfur, with a ring size of 3 to about 12 atoms,or bicyclic ring systems that include a total of about 7 to about 14ring atoms, and optionally substituted with one or more groups asdefined herein under the term “substituted”. A heterocycle can be amonocyclic, bicyclic, or tricyclic group containing one or moreheteroatoms. A heterocycle group also can contain an oxo group (=0) or athioxo (=S) group attached to the ring. Non-limiting examples ofheterocycle groups include 1,3-dihydrobenzofuran, 1,3-dioxolane,1,4-dioxane, 1,4-dithiane, 2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl,imidazolidinyl, imidazolinyl, indolinyl, isochromanyl, isoindolinyl,morpholine, piperazinyl, piperidine, piperidyl, pyrazolidine,pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline, quinuclidine, andthiomorpholine.

The term “heterocycle” can include, by way of example and notlimitation, a monoradical of the heterocycles described in Paquette, LeoA.; Principles of Modern Heterocyclic Chemistry (W.A. Benjamin, NewYork, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistryof Heterocyclic Compounds, A Series of Monographs” (John Wiley & Sons,New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and28; and J. Am. Chem. Soc. 1960, 82, 5566. In one embodiment,“heterocycle” includes a “carbocycle” as defined herein, wherein one ormore (e.g. 1, 2, 3, or 4) carbon atoms have been replaced with aheteroatom (e.g. O, N, or S).

Heterocycles, by way of example and not limitation, includedihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, piperidinyl, 4-piperidonyl,pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,isoindolyl, 3H-indolyl, 1H-indazoly, purinyl, 4H-quinolizinyl,phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl,pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl,pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl,pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl,quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl,benzisoxazolyl, oxindolyl, benzoxazolinyl, isatinoyl, andbis-tetrahydrofuranyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Carbon bonded heterocycles include 2-pyridyl, 3-pyridyl,4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl,6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, and the like.

By way of example and not limitation, nitrogen bonded heterocycles canbe bonded at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole,position 2 of a isoindole, or isoindoline, position 4 of a morpholine,and position 9 of a carbazole, or β-carboline. In one embodiment,nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

The term “amino” refers to -NH₂. The amino group can be optionallysubstituted as defined herein for the term “substituted”, e.g., theamino group can be -NR₂ where R is a group recited in the definition ofsubstituted. For example, the groups -NR₂ can include “alkylamino”wherein at least one R is alkyl and the second R is alkyl or hydrogen,and/or “acylamino” (-N(R)C(=O)R), wherein each R is independentlyhydrogen, alkyl, alkaryl or aryl.

The term “alkaryl” refers to an aryl group substituted with at least onealkyl group, which together form a substituent through a radical oneither the alkyl or the aryl group. The alkyl group of the alkaryl caninclude about 1-8 carbon atoms, either linear or branched. Typicalalkaryl groups include benzyl, 2-phenylethyl, 3-phenylpropyl,4-phenylbuty, 5-phenylpentyl, 6-phenylhexyl, 7-phenylheptyl,8-phenyloctyl, branched alkyl chain derivatives thereof, and naphthaleneversions thereof. The alkaryl can be optionally substituted as describedabove for alkyl groups.

The term “interrupted” indicates that another group is inserted betweentwo adjacent carbon atoms (and the hydrogen atoms to which they areattached (e.g., methyl (CH₃), methylene (CH₂) or methine (CH))) of aparticular carbon chain being referred to in the expression using theterm “interrupted”, provided that each of the indicated atoms' normalvalency is not exceeded and the interruption results in a stablecompound. Suitable groups that can interrupt a carbon chain include,e.g., with one or more non-peroxide oxy (—O—), thio (—S—), imino(—N(H)—), methylene dioxy (—OCH₂O—), carbonyl (—C(═O)—), carboxy(—C(═O)O—), carbonyldioxy (—OC(═O)O—), carboxylato (—OC(═O)—), imine(C═NH), sulfinyl (SO) and sulfonyl (SO₂). Alkyl groups can beinterrupted by one or more (e.g., 1, 2, 3, 4, 5, or about 6) of theaforementioned suitable groups. The site of interruption can also bebetween a carbon atom of an alkyl group and a carbon atom to which thealkyl group is attached.

As to any of the above groups, which contain one or more substituents,it is understood, of course, that such groups do not contain anysubstitution or substitution patterns that are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers arising from thesubstitution of these compounds. It will be appreciated that somecompounds of the present invention may contain asymmetricallysubstituted carbon atoms, and may be isolated in optically active orracemic forms. It is well known in the art how to prepare opticallyactive forms, such as by resolution of racemic forms or by synthesisfrom optically active starting materials. All chiral, diastereomeric,racemic forms and all geometric isomeric forms of a structure are partof this invention. In addition, some compounds of the present inventionmay exist as rotational isomers. In some instances, these rotationalisomers may be separated. Both rotational isomers and a mixture thereofare contemplated by the present invention.

One isomeric form may display superior properties or activity comparedwith another. When required, separation of the racemic material can beachieved by HPLC using a chiral column or by a resolution using aresolving agent such as camphonic chloride as described by Tucker etal., J. Med. Chem., 37: 2437 (1994). A chiral compound may also bedirectly synthesized using a chiral catalyst or a chiral ligand, e.g.Huffman et al., J. Org. Chem., 60: 1590 (1995).

An “effective amount” generally means an amount that provides a desiredeffect, for example, an amount sufficient to bring about a reaction.

As used herein, “contacting” refers to the act of touching, makingcontact, or of bringing to immediate or close proximity, including atthe molecular level, for example, to bring about a chemical reaction orphysical change, e.g., in a solution, cell, or other reaction mixture.

The term “reactive group” refers to an activated ester of a carboxylicacid, an amine, an alcohol, a sulfonyl halide, a mercaptan, a boronate,a phosphoramidite, an isocyanate, a haloacetamide, an aldehyde, anazide, an acyl nitrile, a photoactivateable group or an alkyl halide.

The term “conjugated substance” refers to a covalently bound substancesuch as a surface (e.g. a bead, solid support, resin particle, or anassay plate), biological molecule (e.g., proteins, nucleotides,polynucleotides including DNA and RNA, enzyme substrates, antibodies,nanobodies, polypeptides, polypeptide-based toxins, amino acids, lipids,carbohydrates, haptens, small molecules, drugs, drug compounds,ion-complexing agents, such as metal chelators, microparticles,synthetic or natural polymers, cells, viruses, other fluorescentmolecules or surfaces), or other moieties of interest, e.g. achloroalkane or a cyanobenzothiazole.

A “tracer” is a type of conjugated substance where a dye of the presentinvention is conjugated to a biological molecule as defined above,possibly through a linker.

The term “traceless linker” or “self-immolative linker” refers to alinker wherein cleavage of a conjugated substance from the linkerresults in spontaneous cleavage of the linker from the dye to releasethe unbound dye. Exemplary traceless linkers include:

As would be recognized by one of ordinary skill in the art, furthervariations in linker length and substitution are possible.

Dyes

The invention provides compounds of Formula (Ia) and (Ib):

wherein

R¹ and R¹¹ are independently H or C₁₋₄ alkyl, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R², R⁵, R¹² and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H,SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, and R²⁶ are independently H or C₁₋₆ alkylor one or more of R²⁰ and R²¹, R²¹ and R²², R²² and R²³, R²⁴ and R²⁵,R²⁵ and R²⁶, and R²⁶ and R²³ together form an aryl, heteroaryl,carbocyclic or heterocyclic ring;

R¹ and R² and/or R¹¹ and R¹² may together form a carbocyclic,heterocyclic, aryl or heteroaryl ring;

R⁶⁻¹⁶ are independently H, halo, OH, alkyl, aryl, heteroaryl, CO₂H,SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

each X is independently CHR²³, O, S or NR³⁰; and

R³⁰ is H, C₁₋₄ alkyl or —C(O)C₁₋₄ alkyl.

In some embodiments, the ring formed by R¹ and R² or R¹¹ and R¹² can befrom 3-10 atoms chosen from C, N, O and S. In other embodiments, thering has from 5 to 7 atoms. In certain embodiments, the ring atoms areall carbon. These rings may contain elements of unsaturation as well. Incertain embodiments, these rings are aryl or heteroaryl rings.

In Formula (Ib), R² and R⁵ may together form an aryl or heteroaryl ring.Suitably, the ring is phenyl or thiophenyl. In some embodiments, thering is substituted.

Suitably, X is CH₂. R¹¹ is suitably C₁₋₄ alkyl. In some embodiments, R¹¹is methyl or ethyl. In other embodiments, R¹¹ and R¹² form a 5-7membered carbocyclic ring. Suitably, the ring is an unsubstituted6-membered ring. Suitably, R² and R¹² are H, Cl or OMe. R⁵ and R¹⁵ maybe H.

The invention also provides compounds of formula (IIa) and (IIb):

wherein

R¹ and R¹¹ are independently H or C₁₋₄ alkyl, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R², R³, R⁴, R⁵, R¹², R¹³, R¹⁴ and R¹⁵ are independently H, alkyl, aryl,heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

R²⁰, R²¹, R²², R²³, R²⁴, R²⁵ and R²⁶ are independently H or C₁₋₆ alkylor one or more of

R²⁰ and R²¹, R²¹ and R²², R²² and R²³, R²⁴ and R²⁵, R²⁵ and R²⁶, and R²⁶and R²³ together form an aryl, heteroaryl, carbocyclic or heterocyclicring;

R¹ and R² and/or R¹¹ and R¹² may together form a carbocyclic,heterocyclic, aryl or heteroaryl ring;;

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

each X is independently CHR²³, O, S or NR³⁰; and

R³⁰ is H, C₁₋₄ alkyl or —C(O)C₁₋₄ alkyl.

In some embodiments, the ring formed by R¹ and R² or R¹¹ and R¹² can befrom 3-10 atoms chosen from C, N, O and S. In other embodiments, thering has from 5 to 7 atoms. In certain embodiments, the ring atoms areall carbon. These rings may contain elements of unsaturation as well. Incertain embodiments, the rings may be aryl or heteroaryl rings.

In Formula (IIa), one or more of R² and R³, R⁴ and R⁵, R¹² and R¹³ orR¹⁴ and R¹⁵ can also be joined together with an aryl or heteroaryl ring.In Formula (llb), one or both of R⁴ and R⁵ or R¹⁴ and R¹⁵ may togetherform an aryl or heteroaryl ring. Suitably, the ring is phenyl orthiophenyl. In some embodiments, the ring is substituted.

Suitably, X is CH₂. R¹¹ is suitably C₁₋₄ alkyl. In some embodiments, R¹¹is methyl or ethyl. In other embodiments, R¹¹ and R¹² together form a5-7 membered carbocyclic ring. Suitably, the ring is an unsubstituted6-membered ring. Suitably, R², R⁵, R¹² and R¹⁵ are H.

The invention also provides compounds of formula (IIIa), (IIIb) and(IIIc):

wherein

R¹¹ is independently H or C₁₋₄ alkyl, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R² and R¹⁶ can be independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

R³ and R⁴ are H, alkyl, L-R, L-C_(s), L-CO₂H, L-SO₃H or together form acarbocyclic, aryl, heteroaryl, or heterocyclic ring;

alternatively, R² and R³ and independently R⁴ and R¹⁶ together form acarbocyclic, heterocyclic, aryl or heteroaryl ring;

R⁵, R¹², R¹³, R¹⁴ and R¹⁵ are independently H, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

R²⁰, R²¹, R²² and R²³ are independently H or C₁₋₆ alkyl or one or moreof R²⁰ and R²¹,

R²¹ and R²², R²² and R²³, together form an aryl, heteroaryl, carbocyclicor heterocyclic ring;

R¹¹ and R¹² may together form a carbocyclic, heterocyclic, aryl orheteroaryl ring;

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s);

X is CHR²³, O, S or NR³⁰; and

R³⁰ is H, C₁₋₄ alkyl or —C(O)C₁₋₄ alkyl.

In formulas (IIIa) and (IIIb), R¹⁶ and R⁵ may together form acarbocyclic, heterocyclic, aryl or heteroaryl ring. In formula (IIIc),R¹⁴ and R⁵ and/or R² and R¹³ may together form a carbocyclic,heterocyclic, aryl or heteroaryl ring. Suitably, the ring is a phenyl orthiophenyl. In some embodiments, the ring is substituted.

In some embodiments, the ring formed by R¹¹ and R¹² can be from 3-10atoms chosen from C, N, O and S. In other embodiments, the ring has from5 to 7 atoms. In certain embodiments, the ring atoms are all carbon.These rings may contain elements of unsaturation as well. In certainembodiments, the ring is aryl or heteroaryl.

The invention also provides compounds according to Formula (IV):

wherein

R² and R¹⁶ can be independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R³ and R⁴ are H, alkyl or together form a carbocyclic, aryl, heteroaryl,or heterocyclic ring;

alternatively, R² and R³ and independently R⁴ and R¹⁶ together form acarbocyclic, heterocyclic, aryl or heteroaryl ring;

R⁵ and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

R²² and R²³ are independently H or C₁₋₆ alkyl or together form an aryl,heteroaryl, carbocyclic or heterocyclic ring; and

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s).

The invention further provides compounds according to Formula (V):

wherein

R² and R¹⁶ can be independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R³ and R⁴ are H, alkyl, or together form a carbocyclic, heterocyclic ,aryl or heteroaryl ring;

alternatively, R² and R³ and independently R⁴ and R¹⁶ may together forma carbocyclic, heterocylic, aryl or heteroaryl ring;

R⁵ and R¹⁵ are independently H, alkyl, aryl, heteoaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

R²⁰ and R²¹ are independently H or C₁₋₆ alkyl or together form an aryl,heteroaryl, carbocyclic or heterocyclic ring; and

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s).

The invention additionally provides compounds according to Formula (VI):

wherein

R⁵ and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R²², R²³, R²⁶ and R²⁷ are independently H or C₁₋₆ alkyl or one or moreof R²² and R²³ and R²⁶ and R²⁷ together form an aryl, heteroaryl,carbocyclic or heterocyclic ring;and

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s).

The invention additionally provides compounds according to Formula(VII):

wherein

R⁵ and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R²⁰, R²¹, R²⁴ and R²⁵ are independently H or C₁₋₆ alkyl or one or moreof R²⁰ and R²¹ and R²⁴ and R²⁵ together form an aryl, heteroaryl,carbocyclic or heterocyclic ring; and

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s).

The following statements apply to Formulae (I)-(VII), where appropriate.Suitably, R², R⁵ and R¹⁵ are H. R³ and R⁴ are suitably C₁₋₄ alkyl. Insome embodiments, R³ and R⁴ are methyl or ethyl. R³ and R⁴ may togetherform a heterocycle, such as a piperazine. In other embodiments, R² andR³ and/or R⁴ and R¹⁶ together form a 5-7 membered carbocylic ring. Thering is suitably an unsubstituted 6-membered ring.

R¹⁰ is suitably H, F, Cl, CO₂H or SO₃H. In certain embodiments, R¹⁰ isH. R⁶ and R⁹ are suitably H or halo. In certain embodiments, R⁶ and R⁹may be either Cl or F.

Suitably, one of R⁷ and R⁸ is -L-R, -L-CO₂H or -L-C_(s) and the other isH, Cl, or F.

L is suitably —CO—, —SCH₂CO— or —SO₂—. L may also contain a PEG moiety.In other embodiments, L is a “traceless” or “self-immolative” linker.

Suitably, R is

or —Cl, with SE most preferred.

C_(s) is suitably a chloroalkane of the formulaNHCH₂CH₂(OCH₂CH₂)_(n)(CH₂)₆Cl, with n being 2-6; a nucleoside, forexample

(allylaminodU); an oligonucleotide suitably attached throughallylaminodU; or a cyanobenzothiazole. Suitably, the cyanobenzothiazoleis

wherein Z is O or NH.

Suitable compounds include those shown in FIGS. 1-3. As one of ordinaryskill in the art would understand, any of the combinations betweencolumn 1 and row 1 are feasible. Those that show a compound number havebeen synthesized.

Among other unique properties, compounds of the present inventioncontaining a 7-membered ring show a significant red shift in theexcitation and emission of the dye as compared to prior art compounds.In certain embodiments, the compounds of the present invention haveemission maxima from about 600 nm to about 730 nm. In certainembodiments, the compounds of the present invention have excitationmaxima from about 575 nm to about 675 nm.

Although there is no description of incorporating a fused 7-memberedring in the manner described here into a fluorescent dye in theliterature, two publications (Zachariasse, et al., J. Photochem.Photobiol. A, 1997, 105, 373-383; Saha and Samanta, J. Phys. Chem. A2002, 106, 4763-4771) describe comparisons between incorporating anitrogen involved in dye fluorescence in a six-membered carbocyclic ringand a seven-membered carbocyclic ring. With the dyes described in theseliterature reports, as the ring size is increased, the quantum yielddrops dramatically with increasing solvent polarity, which does notoccur for the dyes of the present invention.

There has also been a discussion of the effect of unfused ring size inrosamine derivatives (Lu and Burgess, J. Org. Chem., 2008, 73,8711-8718). No shift in emission wavelength was observed on varying thering size from a five- to a six-membered ring. The red-shifted emissionof the dyes containing a fused seven-membered ring relative to thosewith six-membered rings while maintaining a high quantum yield in polarsolvents was unanticipated.

Synthesis of Dyes

Compounds described herein may be synthesized and conjugated using avariety of methods. See, e.g., Beija, et al., Chem. Soc. Rev., 2009, 38,2410-2433. Exemplary syntheses are generalized below.

Synthesis of Dye Precursors:

Synthesis of intermediate 3 can be done through formation of an oxime oftetralone 1 followed by a Beckmann rearrangement to give compound 2.Lactam reduction then provides compound 3.

Alkylation of this cyclic amine 3 followed by demethylation providescompound 4.

Alkylation of the nitrogen with bromochloropropane followed by heatinduced cyclization provides a tricyclic amine Demethylation of thisamine provides compound 5. Use of the 5-methoxy tetralone provides theisomeric aminophenol.

Synthesis of Symmetrical Dyes:

Synthesis of symmetrical rhodamines is accomplished through a meltprocedure where compound 3, anhydride 6 and ZnCl₂ are fused togetherwith high heat. When anhydride 6 is unsymmetrical, purification providesboth isomeric dyes, typically in equal amounts. Often, some of thedecarboxylated rosamine is also isolated resulting from heat induceddecarboxylation of compound Ia.

Synthesis of Unsymmetrical Dyes:

Synthesis of unsymmetrical rhodamine dyes is accomplished in a two stepprocedure. In the first step, an aminophenol 3 is reacted with ananhydride 6 in the absence of an acidic catalyst to provide the ketoneadduct 7. As above, if 6 is unsymmetrical the two isomers of 7 can beseparated at this stage.

Compound 7 is then reacted with another aminophenol (or aminonaphthol)to give a dye of formula IIIa. Typical procedure for this reaction is inDMF at 80° C. with catalytic trimethylsilylpolyphosphate.

Conjugation of Halogenated Dyes:

Tetrahalogenated rhodamines (either fluoro or chloro) werefunctionalized by treatment with mercaptoacetic acid in DMF. Thiscarboxylic acid could then be activated as an succidimidyl ester (SE)and linked to biomolecules. Rhodamines with a carboxylic acid on thelower ring were directly activated as an SE and linked to biomolecules.

As can be appreciated by the skilled artisan, alternative methods ofsynthesizing the compounds of the formulae herein will be evident tothose of ordinary skill in the art. Additionally, the various syntheticsteps may be performed in an alternate sequence or order to give thedesired compounds. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Intermediates

The invention further provides compounds of formulae (VIII) and (IX):

wherein

R¹¹ is H or C₁₋₄ alkyl, L-R or L-C_(s);

L is a covalent linkage that is linear or branched, cyclic orheterocyclic saturated or unsaturated, having 1-16 non hydrogen atomssuch that the linkage contains any combination of ester, acid, amine,amide, alcohol, ether, thioether or halide groups or single, double,triple or aromatic carbon-carbon bond;

R is a reactive group;

C_(s) is a conjugated substance;

R¹² and R¹⁵ are independently H, alkyl, aryl, CO₂H, SO₃H, L-CO₂H,L-SO₃H, L-R or L-C_(s);

R²⁰, R²¹, R²² and R²³ are independently H or C₁₋₆ alkyl or one or moreof R²⁰ and R²¹, R²¹ and R²² and R²² and R²³ together form a fused arylring;

R¹¹ and R¹² may be joined together in an optionally substituted ring;

R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl, aryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s);

X is CHR²³, O, S or NR³⁰; and

R³⁰ is H, C₁₄ alkyl or —C(O)C₁₋₄ alkyl.

In some embodiments, the ring formed by R¹¹ and R¹² can be from 3-10atoms chosen from C, N, O and S. These rings may contain elements ofunsaturation as well.

These compounds are useful in the synthesis of compounds of formulae(I)-(VII).

Labeled Biomolecules

Briefly, the dyes of the present invention may be used as labelingagents which allow for the detection of a composition of matter. Thedyes of the present invention can be used to label a broad range ofmolecules, including but not limited to, biomolecules such aspolypeptides, polypeptide-based toxins, amino acids, nucleotides,polynucleotides including DNA and RNA, lipids, carbohydrate, and enzymesubstrates. Additionally, the compounds may be used to label haptens,small molecules, drugs, drug compounds, ion-complexing agents, such asmetal chelators, microparticles, synthetic or natural polymers, cells,viruses, other fluorescent molecules or surfaces. The resulting labeledmolecules may be referred to as conjugates or tracers.

In some aspects, the dyes can be conjugated with a nucleoside,nucleotide or a polynucleotide. The dyes of the invention may beconjugated with nucleoside, nucleotide or polynucleotide in any wayknown to one of ordinary skill in the art such as through aphophoramidite, an activated ester or a reactive platinum complex.

In other aspects, the dyes of the invention can be used to conjugatewith an amino acid, amino acid analog, or polypeptide. In other aspects,the dyes of the invention can be used to conjugate with a smallmolecule, e.g., a drug or drug compound. In some aspects, the conjugatedsmall molecule can be used as a fluorescent tracer.

In some embodiments, the labeled biomolecules may be profluorescentcompounds. A profluorescent compound is one that has a fluorescence thatis reduced as compared to the related dye and contains a substrate foran enzyme of interest. Upon being acted on by the enzyme of interest,the fluorescent dye from the profluorescent compound is released, andthereby fluorescence is generated. A “traceless” or “self-immolative”linker can also be included between the dye and the enzyme substrate.

Exemplary labeled biomolecules include compounds of formulae (XIa),(XIb) and (XIc):

wherein

R¹¹ is H or C₁₋₆ alkyl;

W is a traceless linker or a direct bond;

R is a reactive group;

C_(s) is an enzyme substrate;

R², R³, R⁴ R⁵, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H,alkyl, aryl, CO₂H, SO₃H, L-R, L-C_(s), L-CO₂H, or L-SO₃H;

R¹¹ and R¹² may be joined together in an aryl, heteroaryl, carbocyclicor heterocyclic ring ring;

R⁶⁻⁹ are independently H, F, Cl, Br, I, OH, alkyl, aryl, CO₂H, SO₃H,L-CO₂H, or L-SO₃H; and

X is CH₂, O, S or NH.

In some embodiments, the enzyme substrate is Z-DEVD. In anotherembodiment, the enzyme substrate is Z-AAF-benzyl.

Profluorescent compounds according to formulae (XIa) include:

In other embodiments, the invention provides other profluorescentbiomolecules. In some embodiments, a dye according to the presentinvention is attached through a biomolecule to a quencher. Any quencherthat absorbs in the emission spectrum of the dye can be used. One ofordinary skill in the art would be able to identify such quenchers.Suitable quenchers include Black Hole Quenchers™ and QXL™ quenchers.(Available from Life Technologies).

Exemplary profluorescent compounds containing a quencher include:Quencher-GABA-Pro-Cha-Abu-Smc-His-Ala-Dab(dye)-Ala-Lys-NH2 as an MMP-3substrate where cleavage in the amino acid chain separates the quencherfrom the dye, producing fluorescence. In one embodiment, the dye can beconjugated to the amino acid chain through a carboxylic acid in thelower ring. The dye can also be attached to the amino acid chain atother positions.

Methods of Use

The dyes of the present invention provide an effective tool forcovalently labeling biomolecules for a wide variety of applications.Labeling allows one to study interactions involving biomolecules such asproteins, glycoproteins, nucleic acids and lipids, as well as smallmolecules, e.g., drugs or drug compounds, inorganic chemicals or anycombinations thereof. The interactions may be studied in cell-freebiological systems, in cellular systems or in vivo. Analyzing thevarious interactions is often a significant part of scientific researchand development, drug design, screening and optimization, phylogeneticclassification, genotyping individuals, parental and forensicidentification, environmental studies, diagnosis, prognosis, and/ortreatment of disease conditions.

In some aspects of the invention, the conjugates of the invention areused to label a sample so that the sample can be identified orquantitated. For instance, such conjugates may be added as part of anassay for a biological target analyte or as a detectable tracer elementin a biological or non-biological fluid.

The sample may be obtained directly from biological materials, e.g., awash from a solid material (organic or inorganic), a medium in whichcells have been cultured, a cell lysate, a buffer solution in whichcells have been placed for evaluation, or physiological sources, e.g.,blood, plasma, serum, urine, etc. When the sample comprises cells, thecells are optionally single cells, including microorganisms, or multiplecells associated with other cells in two or three dimensions, includingmulticellular organisms, embryos, tissues, biopsies, filaments,biofilms, and the like. When the sample comprises cells, the cells maybe lysed, e.g., a cell lysate, or whole cells.

Alternatively, the sample is a solid, optionally a smear or scrape or aretentate removed from a liquid or vapor by filtration. In one aspect ofthe invention, the sample is obtained from a biological fluid, includingseparated or unfiltered physiological fluids such as urine,cerebrospinal fluid, blood, lymph fluids, tissue homogenate,interstitial fluid, cell extracts, mucus, saliva, sputum, stool,physiological secretions or other similar fluids. Alternatively, thesample is obtained from an environmental source such as soil, water, orair; or from an industrial source such as taken from a waste stream, awater source, a supply line, or a production lot.

In other embodiments, the sample is present on or in a solid orsemi-solid matrix. In one aspect of the invention, the matrix is amembrane. In other aspects, the matrix is an electrophoretic gel, suchas those used for separating and characterizing nucleic acids orproteins, or a blot prepared by transfer from an electrophoretic gel toa membrane. In other aspects, the matrix is a silicon chip or glassslide, and the analyte of interest has been immobilized on the chip orslide in an array (e.g., the sample comprises proteins or nucleic acidpolymers in a microarray). In yet other aspects, the matrix is amicrowell plate or microfluidic chip, and the sample is analyzed byautomated methods, typically by various methods of high-throughputscreening, such as drug screening.

The dye conjugates are generally utilized by combining the conjugate asdescribed above with the sample of interest under conditions selected toyield a detectable optical response. The sample is then illuminated at awavelength selected to elicit the optical response. Typically, aspecified characteristic of the sample is determined by comparing theoptical response with a standard or expected response.

A detectable optical response means a change in, or occurrence of, anoptical signal that is detectable either by observation orinstrumentally. Typically, the detectable response is a change influorescence, such as a change in the intensity, excitation or emissionwavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The degree and/orlocation of the labeling, compared with a standard or expected response,indicates whether, and to what degree, the sample possesses a givencharacteristic. Some dyes of the invention may exhibit littlefluorescence emission, but are still useful as chromophoric dyes. Suchchromophores are useful as energy acceptors in FRET applications, or tosimply impart the desired color to a sample or portion of a sample.

For biological applications, the dye conjugates are typically used in anaqueous, mostly aqueous or aqueous-miscible solution prepared accordingto methods generally known in the art. The exact concentration of thedye compound is dependent upon the experimental conditions and thedesired results, but typically ranges from about one nanomolar to onemillimolar or more. The optimal concentration may be determined bysystematic variation until satisfactory results, with minimal backgroundfluorescence, are accomplished.

The dye conjugates may be used to label samples containing biologicalcomponents. The sample may comprise heterogeneous mixtures of components(including intact cells, cell extracts, cell lysates, bacteria, viruses,organelles, and mixtures thereof) or a single component or homogeneousgroup of components (e.g., natural or synthetic amino acid, nucleic acidor carbohydrate polymers, or lipid membrane complexes). The dyes aregenerally non-toxic to living cells and other biological componentswithin the concentrations of use.

The dye conjugate may be combined with the sample in a way thatfacilitates contact between the dye conjugate and the sample componentsof interest. Typically, the dye conjugate or a solution containing thedye conjugate is simply added to the sample. Certain dyes of theinvention, e.g., those that are substituted by one or more sulfonic acidmoieties, may be less permeant to membranes of biological cells, butonce inside viable cells are typically well retained. Treatments thatpermeabilize the plasma membrane, such as electroporation, shocktreatments or high extracellular ATP, may be used to introduce selecteddye conjugates into cells. Alternatively, selected dye conjugates can bephysically inserted into cells, e.g., by pressure microinjection, scrapeloading, patch clamp methods, or phagocytosis.

Dyes that incorporate an aliphatic amine or a hydrazine residue may bemicroinjected into cells where they can be fixed in place by aldehydefixatives such as formaldehyde or glutaraldehyde. This property makessuch dyes useful for intracellular applications such as neuronaltracing.

Dyes that possess a lipophilic substituent, such as phospholipids, maynon-covalently incorporate into lipid assemblies, e.g., for use asprobes for membrane structure, or for incorporation in liposomes,lipoproteins, films, plastics, lipophilic microspheres or similarmaterials; or for tracing. Lipophilic dyes are useful as fluorescentprobes of membrane structure.

Chemically reactive dye compounds may covalently attach to acorresponding functional group on a wide variety of materials to formdye conjugates as described above. Using dye compounds to label reactivesites on the surface of cells, in cell membranes, in intracellularcompartments such as organelles, or in the cytoplasm, permits thedetermination of their presence or quantity, accessibility, activity ortheir spatial and temporal distribution in the sample. Photoreactivedyes may be used similarly to photolabel components of the outermembrane of biological cells or as photo-fixable polar tracers forcells.

In some embodiments, chloroalkane-labeled dyes may be used with HaloTag®protein to detect proteins of interest by generating a fusion proteinbetween the HaloTag® protein and the protein of interest. Generally,these fusion proteins are expressed by a cell from a HaloTag® fusionconstruct, and the fusion protein is detected through use of thechloroalkane-labeled dye. This allows detection of protein expression ordetermination of a protein expression time-course, protein localizationor migration. In addition, these proteins can be detected in gels usingthis fluorescent label. The dyes may also be utilized in otherorthogonal labeling systems, such as cutinase, dihydrofolatereductase/trimethoprim SNAP-tag, Clip Tag, Alkyl cytosine transferase(see U.S. Patent Application No. 2012/0237961, which is incorporated byreference herein) and Acyl Carrier Protein (see U.S. Patent ApplicationNo. 2010/0173384, which is incorporated by reference herein). Inaddition, HaloTag is also orthogonal to other labeling chemistries suchas Hsuingen cyclizations (click chemistry), hydrazone and oximeformation and the Staudinger ligation.

Optionally, the sample is washed after labeling to remove residual,excess or unbound dye compound or dye conjugate. The sample isoptionally combined with one or more other solutions in the course oflabeling, including wash solutions, permeabilization and/or fixationsolutions, and solutions containing additional detection reagents. Anadditional detection reagent typically produces a detectable responsedue to the presence of a specific cell component, intracellularsubstance, or cellular condition, according to methods generally knownin the art. When the additional detection reagent has, or yields aproduct with, spectral properties that differ from those of the subjectdye compounds, multi-color applications are possible. This isparticularly useful where the additional detection reagent is a dye ordye conjugate having spectral properties that are detectably distinctfrom those of the labeling dye.

The dye conjugates are used according to methods known in the art, e.g.,use of antibody conjugates in microscopy and immunofluorescent assays;or nucleotide or oligonucleotide conjugates for nucleic acidhybridization assays, nucleic acid amplification reactions, and nucleicacid sequencing (e.g., U.S. Pat. Nos. 5,332,666, 5,171,534, and4,997,928, and WO 94/05688). Dye conjugates of multiple independent dyesof the invention possess utility for multi-color applications.

At any time after or during labeling, the sample is illuminated with awavelength of light selected to give a detectable optical response andobserved with a means for detecting the optical response. Equipment thatis useful for illuminating the dye compounds of the invention includes,but is not limited to, hand-held ultraviolet lamps, mercury arc lamps,xenon lamps, lasers and laser diodes. These illumination sources areoptionally integrated into laser scanners, fluorescence microplatereaders, standard or minifluorometers, or chromatographic detectors.

The optical response is optionally detected by visual inspection or byuse of any of the following devices: CCD cameras, video cameras,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes. Where the sample isexamined using a flow cytometer, examination of the sample optionallyincludes sorting portions of the sample according to their fluorescenceresponse.

Exemplary Methods of Use

i. Detection of Nucleic Acid Polymers

In one embodiment, a dye oligonucleotide conjugate of the presentinvention is combined with a sample that contains, or is thought tocontain, a nucleic acid polymer, incubating the mixture of dyeoligonucleotide conjugate, e.g., probe or primer, and sample for a timesufficient for the oligonucleotide in the conjugate to combine withnucleic acid polymers in the sample to form nucleic acid hybrids(complexes) (i.e., a probe), or to prime nucleic acid synthesis (i.e., aprimer), which may be detected. The characteristics of the labeledmolecules, including the presence, location, intensity, excitation andemission spectra, fluorescence polarization, fluorescence lifetime, andother physical properties of the fluorescent signal, can be used todetect, differentiate, sort, quantitate, sequence and/or analyze aspectsor portions of the sample. The dye conjugates of the invention areoptionally used in conjunction with one or more additional reagents(e.g., detectably different fluorescent reagents) including dyes of thesame class having different spectral properties.

Typically, the dye conjugate is prepared for use by dissolving the dyeconjugate in an aqueous or aqueous miscible solution that is compatiblewith the sample and intended use. For biological samples, where minimalperturbation of cell morphology or physiology is desired, the solutionis selected accordingly.

The labeling solution is made by dissolving the dye conjugate directlyin an aqueous solvent such as water, a buffer solution, such as bufferedsaline (preferably non-phosphate for some viability discriminationapplications), a Tris(hydroxymethyl)-aminomethane (TRIS) buffer(preferably containing EDTA), or a water-miscible organic solvent suchas dimethylsulfoxide (DMSO), dimethylformamide (DMF), or a lower alcoholsuch as methanol or ethanol. The dye conjugate is usually preliminarilydissolved in an organic solvent (e.g., 100% DMSO) at a concentration ofgreater than about 100 times that used in the labeling solution, thendiluted one or more times with an aqueous solvent such as water orbuffer, such that the dye conjugate is present in an effective amount.

Typically labeling solutions for cellular samples have a dyeconcentration greater than 0.1 nM and less than 50 μM, more typicallygreater than 1 nM and less than 10 μM, e.g., between 0.5 and 5 μM.Labeling solutions for electrophoretic gels typically have a dyeconcentration of greater than 0.1 μM and less than 10 μM, more typicallyabout 0.5 to 2 μM. The same holds true when the dye is added to the gelbefore being combined with nucleic acids. Labeling solutions fordetection and quantitation of free nucleic acids in solution typicallyhave a concentration of 0.1 μM to 2 μM. The optimal concentration andcomposition of the labeling solution is determined by the nature of thesample (including physical, biological, biochemical and physiologicalproperties), the nature of the dye-sample interaction (including thetransport rate of the dye to the site of the nucleic acids), and thenature of the analysis being performed, and can be determined accordingto standard procedures.

The nucleic acid in the sample may be DNA or RNA, or a mixture or ahybrid thereof. Any DNA is optionally single-, double-, triple-, orquadruple-stranded DNA; any RNA is optionally single stranded (“ss”) ordouble stranded (“ds”). The nucleic acid may be a natural polymer(biological in origin) or a synthetic polymer (modified or preparedartificially). The nucleic acid polymer (for instance, one containing atleast 8 bases or base pairs) may be present as nucleic acid fragments,oligonucleotides, or larger nucleic acid polymers with secondary ortertiary structure. The nucleic acid is optionally present in acondensed phase such as a chromosome. The nucleic acid polymeroptionally contains one or more modified bases or links or containslabels that are non-covalently or covalently attached. For example, themodified base can be a naturally occurring modified base such as Ψ(pseudouridine) in tRNA, 5-methylcytosine, 6-methylaminopurine,6-dimethylaminopurine, 1-methylguanine, 2-methylamino-6-hydroxypurine,2-dimethylamino-6-hydroxypurine, 5-amino-DU, isoC, isoG, or other knownminor bases (see, e.g., Davidson, The Biochemistry Of The Nucleic Acids(1976)) or is synthetically altered to contain an unusual linker such asmorpholine derivatized phosphates (AntiVirals, Inc., Corvallis, Oreg.),or peptide nucleic acids such as N-(2-aminoethyl)glycine units (Wittunget al., Nature, 368:561 (1994)) or contain a simple reactive functionalgroup (<10 carbons) that is an aliphatic amine, carboxylic acid,alcohol, thiol or hydrazine, or contain a fluorescent label or otherhapten, such as inosine, bromodeoxyuridine, iododeoxyuridine, biotin,digoxigenin, 2,4-dinitrophenyl, where the label is originally attachedon the nucleotide (e.g., CHROMATIDE™ labeled nucleotides, MolecularProbes, Eugene, Oreg.) or located on the 3′ or 5′ end of a nucleic acidpolymer, or ligands non-covalently attached to the nucleic acids. Thesensitivity of the dyes for nucleic acid polymers containing primarilymodified bases and links may be diminished by interference with thebinding mode. Some embodiments of the dyes may inhibit non-specificnuclease activity but not restriction endonuclease activity at certaindye:base pair ratios.

The sample that contains a nucleic acid is optionally a biologicalstructure (i.e., an organism or a discrete unit of an organism), or asolution (including solutions that contain biological structures), or asolid or semi-solid material. Consequently, the nucleic acid isoptionally free in solution, immobilized in or on a solid or semi-solidmaterial, extracted from a biological structure (e.g., from lysed cells,tissues, organisms or organelles), or remains enclosed within abiological structure. In order for the nucleic acids to bind to thedyes, it is necessary that the nucleic acids be in an aqueousenvironment to allow contact with the dye, even if the nucleic acids arenot enclosed in a biological structure.

The biological structure that contains the nucleic acid is optionally acell or tissue, for example, where the nucleic acid is present in a cellor interstitial space, as a prokaryote or eukaryote microorganism, or asa virus, viroid, chromosome or organelle. Alternatively, the biologicalstructure may not be contained in a tissue or cell and is present eitheras a virus or as a microorganism or other cell, or is present as acellular component removed from its parent cell (e.g., a plasmid orchromosome, or a mitochondrion or nucleus or other organelle).Typically, the biological structure is an organelle, chromosome or cellthat is optionally contained within a eukaryote cell. The cell presentinside a eukaryote cell is typically a parasite or other infectiousagent such as a virus, bacterium, protozoa, mycoplasma or mycobacterium.When the nucleic acid is contained in a biological structure that is acell, the cells are viable or dead cells or a mixture thereof, i.e., theintegrity of the cell membrane is optionally intact or disrupted bynatural (autolytic), mechanical or chemical means or by environmentalmeans such as changes in temperature or pressure. Alternatively, thecells are blebbing or undergoing apoptosis or in a cycle of growth orcell division.

When the nucleic acid is present in a solution, the sample solution canvary to contain one of purified or synthetic nucleic acids such asoligonucleotides to crude mixtures such as cell extracts or homogenatesor other biological fluids, or dilute solutions from biological,industrial, or environmental sources. In some cases, it is desirable toseparate the nucleic acids from a mixture of biomolecules or fluids inthe solution prior to combination with the dye. Numerous techniquesexist for separation and purification of nucleic acids from generallycrude mixtures with other proteins or other biological molecules. Theseinclude such means as chromatographic techniques and electrophoretictechniques using a variety of supports or solutions or in a flowingstream. Alternatively, mixtures of nucleic acids may be treated withRNase or DNase so the nucleic acid polymer is not degraded in thepresence of the nuclease can be discriminated from degradation productsusing the subject dyes.

The relatively low toxicity of the dyes of the invention to livingsystems generally enables the examination of nucleic acids in livingsamples with little or no damage caused by the dye itself. For use withintact cells or samples in a gel, more permeant dyes may be employed,although some cells readily take up dyes that have been shown to beimpermeant to other cells by means other than passive diffusion acrosscell-membranes, e.g., by phagocytosis or other types of ingestion. Thesedyes can be used in standard gel-based applications. The photostability,toxicity, binding affinity, quantum yield, and fluorescence enhancementof dyes are determined according to standard methods known in the art.

In one embodiment, a dye oligonucleotide conjugate, e.g., probe orprimer, is employed in methods and kits for the identification ofalleles in a physiological sample. In one embodiment, an appropriate setof loci, primers, and amplification protocols is selected to generateamplified alleles from multiple co-amplified loci which, in oneembodiment, do not overlap in size or which are labeled in a way whichenables one to differentiate between the alleles from different lociwhich overlap in size. In addition, this method contemplates theselection of short tandem repeat (STR) loci which are compatible for usewith a single amplification protocol. Successful combinations can begenerated by trial and error of locus combinations, by selection ofprimer pair sequences, and by adjustment of primer concentrations toidentify an equilibrium in which all included loci may be amplified. Thenumber of loci which may be amplified in a multiplex amplificationreaction step may be from 2 to 50, or any integer between 2 and 50, e.g.16, 17, 18, 21, 23, or 26, so long as the reaction produces amplifiedalleles that can be identified. In one embodiment, the amplifiedfragments are less than 500 by in length.

Synthesis of the primers used in the present method can be conductedusing any standard procedure for oligonucleotide synthesis known tothose skilled in the art. At least one primer for each locus iscovalently attached to a different dye label.

Samples of genomic DNA can be prepared for use in the method of thisinvention using any method of DNA preparation which is compatible withthe amplification of DNA. Many such methods are known by those skilledin the art. When the at least one DNA sample to be analyzed is humangenomic DNA, the DNA may be prepared from samples, selected from thegroup consisting of tissue, blood, semen, vaginal cells, hair, saliva,urine, bone, buccal samples, amniotic fluid containing placental cellsor fetal cells, chorionic villus, and mixtures of any of the sampleslisted above.

Once a sample of genomic DNA is prepared, the targeted loci can beco-amplified in the multiplex amplification step. Any one of a number ofdifferent amplification methods can be used to amplify the loci,including, but not limited to, polymerase chain reaction (PCR),transcription based amplification and strand displacement amplification(SDA). In one embodiment, the DNA sample is subjected to PCRamplification using primer pairs specific to each locus in the set.

At least one primer for each locus can be covalently attached to a dyelabel, one of which comprises a dye of the present invention. Theprimers and dyes attached thereto are selected for use in the multiplexamplification reaction such that the alleles amplified using primers foreach locus labeled with one color do not overlap with the alleles of theother loci in the set co-amplified therein using primers labeled withthe same color, when the alleles are separated, e.g., by gel orcapillary electrophoresis. Fluorescent labels suitable for attachment toprimers for use in the present invention are commercially available.See, e.g. fluorescein and carboxy-tetramethylrhodamine labels and theirchemical derivatives from PE Biosystems and Molecular Probes. In oneembodiment, at least three different labels are used to label thedifferent primers used in the multiplex amplification reaction. When asize marker is included to evaluate the multiplex reaction, the primersused to prepare the size marker may be labeled with a different labelfrom the primers used to amplify the loci of interest in the reaction.

Once a set of amplified alleles is produced from the multiplexamplification step, the amplified alleles are evaluated. The evaluationstep of this method can be accomplished by any one of a number ofdifferent means. Electrophoresis may be used to separate the products ofthe multiplex amplification reaction, e.g., capillary electrophoresis ordenaturing polyacrylamide gel electrophoresis. Gel preparation andelectrophoresis procedures and conditions for suitable for use in theevaluating step are known to the art. Separation of DNA fragments in adenaturing polyacrylamide gel and in capillary electrophoresis occursbased primarily on fragment size.

Once the amplified alleles are separated, the alleles and any other DNAin the gel or capillary (e.g., DNA size markers or an allelic ladder)can then be visualized and analyzed. In one embodiment, the method fordetection of multiplexes containing numerous loci is fluorescence, whereprimers for each locus in the multiplexing reaction is followed bydetection of the labeled products employing a fluorometric detector.

ii. Cell Imaging

Fluorescently-labeled biomolecules have proven extremely useful asreporters for gene expression studies in both cultured cells and entireanimals. For example, in living cells, fluorescently-labeled proteinsare most commonly utilized to track the localization and dynamics ofproteins, organelles, and other cellular compartments, as well as atracer of intracellular protein trafficking. Quantitative imaging oflabeled biomolecules according to the present invention is readilyaccomplished with a variety of techniques, including widefield,confocal, and multiphoton microscopy and provides a unique window forexposing the intricacies of cellular structure and function. Among otherthings, the dyes of the present invention can be used to imagesubcellular protein translocation, to detect protein-protein andprotein-DNA complexes, and to determine protein expression, localizationand activity state.

In one embodiment, the cell is contacted with a labeled biomoleculeaccording to the present invention, and fluorescence is detected. Forexample, a protein can be labeled with a dye of the present inventionand used to bind to a cell surface receptor. In this example, thelocation of the cell surface receptor can be detected.

Alternatively, fluorescent dyes according to the present invention canbe used in a modular protein tagging system such as HaloTag® protein(Promega, Madison WI). In this type of system, the protein tag is amodified haloalkane dehalogenase designed to covalently bind to asynthetic ligand which has a chloroalkane linker attached to afluorescent dye according to the present invention.

iii Enzyme Assays

The dyes of the present invention can also be conjugated to enzymesubstrates and used to detect the presence and/or activity of an enzymein a sample. Thus, the invention provides a method of detecting anenzyme in a sample. In one embodiment, a sample suspected of containingan enzyme is contacted with a labeled biomolecule which is aprofluorescent form of a dye of the present invention and a substratefor the enzyme; and fluorescence is detected.

iv. Other Uses

The fluorescent dyes of the present invention can be used in othertechniques known to those skilled in the art. For example, the dyes maybe used in antibody staining, in studies of organometallic catalysis inliving cells, in biomedical imaging, in in vivo detection of smallmolecules, thiol-reactive probes, biotin and hapten derivatives, nucleicacid and protein analysis, for probing cellular structure (includingcytoskeletal proteins, organelles, lipids and membranes and asfluorescent tracers of cell morphology and fluid flow), and for probingcellular function (including cell viability, cell proliferation,endocytosis, receptors, ion channels, signal transduction, ROS, variouscations, and membrane potential). The dyes may also be used to detectbiological phenomena using FRET or BRET. See, e.g. “The MolecularProbes® Handbook” (www.invitrogen.com) for a description of various usesfor the dyes of the present invention.

The dyes may also be used to detect biological phenomena usingfluorescent resonance energy transfer FRET or bioluminescence resonanceenergy transfer (BRET). See, e.g. “The Molecular Probes® Handbook”(www.invitrogen.com) for a description of various uses for the dyes ofthe present invention. In some embodiments, the dyes disclosed hereincan be used as BRET acceptors. If a dye described herein is broughtwithin the energy transfer radius (e.g., typically <10 nm) of aluciferase and is in the correct orientation, radiationless energytransfer will occur, and the dye will emit light at its normal emission.There are many methods known for bringing the dye close to a luciferase,e.g., small molecules or quantum dots (Xia, Z and Rao, J. 2009. Curr.Opin. Biotech 20: 37-44), and these methods enable one to learnsomething about a biological system of interest.

In some embodiments, a dye disclosed herein may be conjugated to a tagsuch as a chloroalkane (see U.S. Pat. No. 7,867,726). This conjugationcauses the dye to be strongly associated to a protein of interest in acell or cell lysate if that protein is expressed as a HaloTag® fusion.Such a fusion of a protein of interest and HaloTag protein would becovalently labeled with the dye conjugate. There are many other methodsof associating a dye disclosed herein with a specific protein, such asconjugating the dye to a specific antibody. Another protein of interestin the cell or cell lysate can then be fused to a luciferase such as afirefly luciferase or Oplophorus luciferase, e.g., NanoLuc™ luciferasefrom Promega Corporation (see U.S. 20100281552 and U.S. 13/287,986).Upon addition of a luciferin substrate, BRET would be observed if thefirst and second proteins of interest interact at a defined distance(e.g., typically <10 nm). Such a system allows one to study theinteraction of two specific proteins under various conditions, e.g.,inside of a living cell.

In some embodiments, the use of a dye disclosed herein in FRET or BRETcan be used to ascertain the biological interaction of any two materialsof interest such as nucleic acids, lipids, polysaccharides, antibodies,small molecules, e.g. drugs or drug compounds, etc. In some embodiments,the interaction of a small molecule with a protein occurs inside of aliving cell. In some embodiments, a dye disclosed herein may beconjugated to a small molecule, e.g., a tracer, that binds to a proteinof interest in such a way that the molecule/protein interaction is notdisturbed by the dye conjugation. If the protein of interest isexpressed as a luciferase fusion as described above, the interaction ofthe small molecule with the protein can be measured inside a live cell.Such an assay may also be used to investigate the binding of apromiscuous small molecule with only a single protein regardless of howmany other proteins the particular small molecule may bind.

In some embodiments, the reactive dyes of the present invention may beused to label a protein(s) or peptide(s) for quantification of proteininteractions in situ. In some aspects, a dye label could be attached tothe target protein or peptide of interest using the reactivecyanobenzothiazole (CBT) labeling chemistry (see U.S. Patent ApplicationNo. 2009/0263843, which is incorporated by reference herein). The CBTlabeling chemistry requires a free, N-terminal cysteine residue on thetarget protein or peptide, which can be generated in situ or in abiochemical format, e.g., by site-specific proteolytic cleavage (e.g.cleavage of an N-terminal reporter such as HaloTag from a C-terminaltarget protein or peptide). Once proteolysis has occurred and anN-terminal cysteine residue generated, the reactive CBT labeling methodcan be used to generate a single dye label on the target protein orpeptide of interest. This method could also be used for site-specificlabeling of receptor ligands (e.g. cytokines or peptide ligands). Whenthe dye-labeled ligand is bound in close proximity to a cell surfacereceptor labeled with a suitable energy donor (e.g. luciferase forbioluminescence resonance energy transfer (BRET) or a short-wavelengthfluorophore for Forster resonance energy transfer (FRET)), energytransfer can occur between the excited state donor and fluorescent dyeacceptor, leading to an increase in emission from the conjugated dye.Energy transfer could then be used to quantify the interaction of theprotein/peptide that has been labeled with the CBT-dye with thedonor-labeled receptor of interest. This labeling method may becompatible with purified components, e.g., purified protein or peptide,or in more complex samples including whole cells or cell lysates.Furthermore, this labeling method may be useful for testing the affinityof unlabeled proteins/peptides for a receptor of interest by competitivedisplacement of the ligand-receptor complex generating the BRET signal.

Kits

One aspect of the invention is the formulation of kits that facilitatethe practice of various assays using any of the dyes of the invention,as described above. The kits of the invention typically comprise acolored or fluorescent dye of the invention, either present as achemically reactive label useful for preparing dye-conjugates or presentas a dye-conjugate where the conjugated substance is a specific bindingpair member, or, for instance, a nucleoside, nucleotide,oligonucleotide, polynucleotide, peptide, protein or small molecule,e.g. drug or drug compound. The kit optionally further comprises one ormore buffering agents, typically present as an aqueous solution. Thekits of the invention optionally further comprise additional detectionreagents, a purification medium for purifying the resulting labeledsubstance, luminescence standards, enzymes, enzyme inhibitors, organicsolvent, constructs for expression of fusion proteins, e.g., fusionproteins comprising a luciferase or HaloTag® protein fused to a proteinor target of interest, fusion proteins, or instructions for carrying outan assay of the invention.

In some embodiments, a kit of the invention includes one or morelocus-specific primers. Instructions for use optionally may be included.Other optional kit components may include an allelic ladder directed toeach of the specified loci, a sufficient quantity of enzyme foramplification, amplification buffer to facilitate the amplification,loading solution for preparation of the amplified material forelectrophoresis, genomic DNA as a template control, a size marker toinsure that materials migrate as anticipated in the separation medium,and a protocol and manual to educate the user and to limit error in use.The amounts of the various reagents in the kits also can be varieddepending upon a number of factors, such as the optimum sensitivity ofthe process. It is within the scope of this invention to provide testkits for use in manual applications or test kits for use with automateddetectors or analyzers.

In other embodiments, the kit also includes a genetically-modified cellor a vector for gene fusion, e.g., fusion comprising a luciferase orHaloTag® protein fused to a protein or target of interest. Instructionsfor use optionally may be included.

The following Examples are intended to illustrate the invention aboveand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples may suggest other ways inwhich the present invention could be practiced. It should be understoodthat variations and modifications may be made while remaining within thescope of the invention.

EXAMPLES Example 1 7-methoxytetralone oxime

To a solution of 7-methoxytetralone (15 g) in MeOH (175 mL), NH₂OH (50%in H₂O, 15.7 mL) and AcOH (4 mL) was added. After stirring for 1 hour,the solution was concentrated until a solid began to appear. Thereaction was poured into dilute aqueous NaHCO₃ (500 mL), and theresulting solid was filtered to provide the title compound (17.8 g): 1HNMR (DMSO-d6) δ 11.08 (s, 1H), 7.35 (s, 1H), 7.08 (d, 1H), 6.84 (d, 1H),3.71 (s, 3H), 2.62-2.49 (m, 4H), 1.77-1.64 (m, 2H).

Example 2 8-methoxy-4,5-dihydro-1H-benzo[b]azepin-2(3H)-one

To a solution of 7-methoxytetralone oxime (17.8 g) in pyridine (400 mL),p-toluenesulfonyl chloride (26.6 g) was added portion wise. Afterstirring for 18 hours, the reaction was poured into HCl (3M, 1L), andthe resulting solid was filtered to provide the crude tosylate (32.4 g).To this orange solid, EtOH (750 mL) and NaOAc (77.0 g in 750 mL H₂O) wasadded, and the resulting suspension was heated to reflux. Afterrefluxing for 16 hours, the heat was removed, and the solution wasconcentrated until solid began to appear. After cooling, filtered offthe resulting white solid to provide the title compound (11.2 g): 1H NMR(DMSO-d6) δ 9.43 (s, 1H); 7.12 (d, 1H), 6.64 (dd, 1H), 6.52 (d, 1H),3.69 (s, 3H), 2.56 (t, 2H), 2.12 (t, 2H), 2.07-1.98 (m, 2H).

Example 3 8-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine

To a solution of 8-methoxy-4,5-dihydro-1H-benzo[b]azepin-2(3H)-one (2 g)in THF (100 mL), lithium aluminum hydride (0.79 g) was added, and thereaction heated to reflux. After stirring for 1 hour, the heat wasremoved. Water (6 mL) was added followed by NaOH (10%, 15 mL), theflocculated solids removed by filtration, and the eluent concentrated toprovide the title compound (1.9 g) as a pale brown oil: 1H NMR (DMSO-d6)δ 6.88 (d, 1H); 6.37 (d, 1H), 6.22 (dd, 1H), 5.14 (s, 1H), 3.62 (s, 3H),2.90-2.85 (m, 2H), 2.57-2.53 (m, 2H), 1.67-1.59 (m, 2H), 1.52-1.45 (m,2H).

Example 4 8-methoxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine

To a solution of 8-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine (0.5 g)in acetonitrile (25 mL), iodoethane (0.45 mL) and K₂CO₃ (1.2 g) wasadded, and the reaction heated to reflux. After stirring for 18 hours,the heat was removed, and the reaction was concentrated. The resultingresidue was partitioned between water (30 mL) and EtOAc (25 mL), thelayers separated, and the organic layer washed with brine, dried(Na₂SO₄) and concentrated to provide the title compound (0.54 g) as aclear oil: 1H NMR (DMSO-d6) δ 6.93 (d, 1H); 6.39 (d, 1H), 6.33 (dd, 1H),3.67 (s, 3H), 3.07 (q, 2H), 2.85-2.82 (m, 2H), 2.62-2.58 (m, 2H),1.66-1.59 (m, 2H), 1.51-1.43 (m, 2H), 1.10 (t, 3H).

Example 5 8-hydroxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine

To a solution of 8-methoxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine(0.54 g) in CH₂Cl₂ (25 mL) cooled to -78° C., BBr₃ (1.24 mL) was added,and the reaction allowed to gradually warm to -20° C. After stirring for3 hours, the reaction was quenched with MeOH, allowed to warm to roomtemperature, and then concentrated. The residue was dissolved in HCl(1M, 40 mL) and stirred for 1 hour. This solution was brought to pH 12with K₂CO₃ (sat. aq.) and extracted with EtOAc (40 mL). The organiclayer was washed with brine, dried (Na₂SO₄) and concentrated. The crudeproduct was purified by silica gel chromatography (gradient of EtOAc inheptane) to provide the title compound (0.37 g) as a clear oil: 1H NMR(DMSO-d6) δ 8.88 (s, 1H), 6.79 (d, 1H); 6.29 (d, 1H), 6.16 (dd, 1H),3.02 (q, 2H), 2.82-2.79 (m, 2H), 2.57-2.53 (m, 2H), 1.65-1.57 (m, 2H),1.48-1.42 (m, 2H), 1.09 (t, 3H); MS expected 192 (C₁₂H₁₈NO, M+1), found192.

Example 6 Bis(ethylazepino)tetrachlororhodamine (PBI 3737)

A mixture of 8-hydroxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine (50mg), tetrachlorophthalic anhydride (52 mg) and ZnCl₂ (36 mg) was heatedto approximately 250° C. for 2 minutes. The residue was suspended inCH₂Cl₂/MeOH (1/1, 20 mL), filtered and concentrated. The crude dye waspurified by preparative HPLC (gradient of ACN in 0.1% TFA in H₂O) toprovide the title compound (1 mg) as a blue solid: MS expected 632(C₃₂H₃₀Cl₄N₂O₃ ⁺, M⁺), found 632; λmaxAbs=595 nm (MeOH), λmaxEm=619 nm(MeOH).

Example 7 Synthesis of Additional Compounds

The following compounds were synthesized in the same manner as PBI 3737using the appropriate phenol and phthalic anhydride. In some cases, therosamine resulting from decarboxylation of the rhodamine was alsoisolated.

PBI Structure Number MS λ_(max)Abs λ_(max)Em

3738 656 608 629

3761 613 610 633

3739 562 583 604

3740 562 585 611

3736 661 594 621

3760 617 596 626

3762 631 601 621

3763 631 602 621

3768 590 607 629

631

3970 631 604 617

Example 8 11-methoxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline

To a solution of 8-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine (0.25g) in bromochloropropane (2 mL), Na₂CO₃ (0.6 g) was added, and thereaction heated to reflux. After stirring for 18 hours, the heat wasremoved, the reaction partitioned between water (30 mL) and ether (25mL), the layers separated, and the organic layer washed with brine,dried (Na₂SO₄) and concentrated. The crude product was purified bysilica gel chromatography (gradient of EtOAc in heptane) to provide thetitle compound (0.27 g) as a clear oil: 1H NMR (DMSO-d6) δ 6.82 (d, 1H);6.36 (d, 1H), 3.68 (s, 3H), 3.04-3.00 (m, 2H), 2.89-2.85 (m, 2H),2.60-2.57 (m, 2H), 2.53-2.49 (m, 2H), 1.70-1.61 (m, 4H), 1.48-1.40 (m,2H); MS expected 218 (C₁₄H₂₀NO, M+1), found 218.

Example 9 11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline

The title compound was synthesized in a similar manner as8-hydroxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine from11-methoxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline: 1H NMR(DMSO-d6) δ 8.82 (s, 1H), 6.64 (d, 1H); 6.22 (d, 1H), 3.03-2.99 (m, 2H),2.87-2.83 (m, 2H), 2.55-2.50 (m, 2H), 2.53-2.49 (m, 2H), 1.68-1.61 (m,4H), 1.45-1.37 (m, 2H); MS expected 204 (C₁₃H₁₈NO, M+1), found 204.

Example 10 8-methoxy-5-methyl-2,3-dihydro-1H-benzo[b]azepine

The title compound was synthesized following the procedure forsynthesizing 8-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine using8-methoxy-5-methyl-1H-benzo[b]azepin-2(3H)-one (Aust. J. Chem. 1978, 31,2031-2037) as starting material: 1H NMR (DMSO-d6) δ 7.28 (d, 1H), 6.46(dd, 1H); 6.28 (d, 1H), 5.89 (t, 1H), 5.30 (s, 1H), 3.77 (s, 3H), 3.40(t, 2H), 2.41 (q, 2H), 2.14 (d, 3H); MS expected 190 (C₁₂H₁₆NO, M+1),found 190.

Example 11 8-hydroxy-5-methyl-2,3-dihydro-1-ethylbenzo[b]azepine

The title compound was synthesized from8-methoxy-5-methyl-2,3-dihydro-1H-benzo[b]azepine following thealkylation procedure for8-methoxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine followed by thedemethylation procedure used to synthesize8-hydroxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine: 1H NMR (DMSO-d6) δ9.15 (s, 1H), 7.05 (d, 1H), 6.31-6.25 (m, 2H), 5.80 (t, 1H), 5.30 (s,1H), 3.12-3.03 (m, 4H), 2.17 (q, 2H), 1.97 (s, 3H), 1.09 (t, 3H); MSexpected 203 (C₁₃H₁₈NO, M+1), found 203.

Example 12 8-hydroxy-5-methyl-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine

A suspension of 8-hydroxy-5-methyl-2,3-dihydro-1-ethylbenzo[b]azepine(0.14 g) and Pd/C (10 mg) in MeOH (10 mL) was purged with H₂ and thenstirred under 1 atm H₂ for 3 hours. The reaction was then filtered overCelite, the eluent concentrated, and the crude reaction purified oversilica gel (gradient of EtOAc in heptane) to provide the title compound(0.09 g) as a clear oil: 1H NMR (CDCl3) δ 7.26 (s, 1H), 6.97 (d, 1H),6.46-6.33 (m, 2H), 3.23-3.02 (m, 4H), 2.79-2.67 (m, 2H), 1.81-1.53 (m,3H), 1.29 (d, 3H), 1.19 (t, 3H); MS expected 206 (C₁₃H₂₀NO, M+1), found206.

Example 13 9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline

The title compound was synthesized in a similar manner as11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone startingfrom 5-methoxytetralone: 1H NMR (DMSO-d6) δ 8.69 (s, 1H), 6.55 (d, 1H);6.27 (d, 1H), 3.05-3.02 (m, 2H), 2.92-2.89 (m, 2H), 2.68-2.64 (m, 2H),2.54 (t, 2H), 1.69-1.60 (m, 4H), 1.45-1.37 (m, 2H); MS expected 204(C₁₃H₁₈NO, M+1), found 204.

Example 142,3,4,5-tetrachloro-6-(9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline-10-carbonyl)benzoicacid

To a solution of9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone (30 mg) indichlorobenzene (1 mL), tetrachlorophthalic anhydride (0.13 mL) wasadded. After stirring at reflux for 2 hours, the solvent was removed,and the resulting crude product purified by preparative HPLC (gradientof ACN in 0.1% TFA in H₂O) to provide the title compound (20 mg) as agreen solid: MS expected 490 (C₂₁H₁₈Cl₄NO₄ ⁺, M+), found 490.

Example 15 Synthesis of Additional Compounds.

The following compounds were synthesized in the same manner as2,3,4,5-tetrachloro-6-(9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,14j]quinoline-10-carbonyl)benzoicacid using the appropriate phenol and phthalic anhydride:

Structure MS

489

396

396

464

464

424

Example 16 Bis(azepinopiperidino)-tetrachlororhodamine (PBI 3861)

To a solution of2,3,4,5-tetrachloro-6-(9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline-10-carbonyl)benzoicacid (20 mg) and9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-iMuinolone (14 mg) inDMF (1 mL), trimethylsilylpolyphosphate (0.25 mL) was added. Afterstirring at 80° C. for 1 hour, water (1 mL) was added, and the resultingsolution purified by preparative HPLC (gradient of ACN in 0.1% TFA inH₂O) to provide the title compound (10 mg) as a blue solid: MS expected656 (C₃₄H₃₀Cl₄N₂O₃ ⁺, M+), found 656; λmaxAbs=605 nm (MeOH),λmaxEm=620nm (MeOH).

Example 17 Synthesis of Additional Compounds.

The following compounds were synthesized in the same manner asbis(azepinopiperidino)-tetrachlororhodamine using the appropriate phenoland ketophenol from Example 14 or 15.

PBI Structure Number MS λ_(max)Abs λ_(max)Em

4273 694 647 713

4302 693 668 741

4335 641 632 690

4351 548 602 662

4352 670 614 632

4379 591 590 612

4382 497 564 596

4428 566 583 604

4464 641 611 644

4484 667 614 646

4490 617 592 616

4496 643 601 621

4497 616 598 637

4553 682 619 637

575 602 642

617 617 638

4624 497 571 601

Example 18 Bis(piperidineazepino)-trichlororhodamine mercaptoacetic Acid(PBI 3769)

To a solution of bis(piperidineazepino)-tetrachlororhodamine (PBI 3738,0.60 g) and diisopropylethylamine (0.32 mL) in DMF (10 mL),mercaptoacetic acid (0.13 mL) was added. After stirring for 2 hours, theresulting product was purified by preparative HPLC (gradient of ACN in0.1% TFA in H₂O) to provide the title compound (0.35 g) as a blue solid:MS expected 712 (C₃₆H₃₃Cl₃N₂O₅S⁺, M+), found 712; λmaxAbs=606 nm (MeOH),λmaxEm=627 nm (MeOH).

Example 19 Synthesis of Additional Compounds.

The following compounds were synthesized in the same manner as PBI 3769using the appropriate halorhodamine:

PBI Structure Number MS λ_(max)Abs λ_(max)Em

662 610 633

3865 637 617 642

749

647 589 613

4577 699 600 620

4555 727 610 629

4559 647 604 645

4568 689 613 633

4681 721 614 652

Example 20 Bis(piperidinoazepino)-pentafluororosamine (PBI 3860)

A solution of pentafluorobenzaldehyde (40 mg) and11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone (50 mg)in H₂SO₄ (60% aqueous, 2 mL) was stirred at 150° C. for 10 minutes. Theresulting solution was purified by preparative HPLC (gradient of ACN in0.1% TFA in H₂O) to provide the title compound (20 mg) as a blue solid:MS expected 565 (C₃₃H₃₀F₅N₂O⁺, M+), found 565; λ_(max)Abs=620 nm (MeOH),λ_(max)Em=642 nm (MeOH).

Example 21Bis(piperidineazepino)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-carbamoyl)rhodamine(PBI 3781)

To a solution of bis(piperidineazepino)-6-carboxyrhodamine (10 mg) anddiisopropylethylamine (0.02 mL) in DMF (1 mL), TSTU (8 mg) was added.After stirring for 15 minutes,2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine HCl (7 mg) was added, whichwas synthesized according to the procedure described in H. Benink, M.McDougall, D. Klaubert, G. Los, BioTechniques 2009, 47, 769-774 (whichis incorporated by reference herein). After stirring another 30 minutes,the reaction mixture was purified by preparative HPLC (gradient of ACNin 0.1% TFA in H₂O) to provide the title compound (1 mg) as a bluesolid: MS expected 769 (C₄₅H₅₅ClN₃O₆ ⁺, M+), found 769.

Example 22 Synthesis of Additional Compounds

The following compounds were synthesized in the same manner as PBI 3781using the appropriate dye carboxylic acid and amine.

PBI Structure Number MS

3780 868

3782 838

3783 917

3905 926

3906 838

1280

3954 838

4356 953

4357 1042

4830 769

4839 857

4840 945

Example 23 Bis(piperidineazepino)trichlororhodamine acetoallylaminodU5′-DMT 3′-phosphoramidite (PBI 3885)

Solid bis(piperidineazepino)trichlororhodamine acetoallylaminodU 5′-DMT(1.0 g) was flushed with N₂ and dissolved in dry CH₂Cl₂ (10 mL). To thissolution, 5-ethylthiotetrazole (30 mg) followed by2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite (0.31 mL) wasadded. After stirring for 90 minutes, the reaction mixture was added toheptane dropwise. The slurry was stirred for 5 minutes and filtered toprovide the title compound (1.0 g) as a blue solid: MS expected 1480(C₇₈H₈₄Cl₃N₇O₁₂PS+, M+), found 1480.

Example 24 Bis(piperidineazepino)-trichlororhodamine mercaptoacetic AcidSE (PBI 4574)

To a solution of bis(piperidineazepino)-trichlororhodaminemercaptoacetic acid (10 mg) and diisopropylethylamine (0.02 mL) inCH₂Cl₂ (0.5 mL), 2-succimido-1,1,3,3-tetramethyluroniumtetrafluoroborate (TSTU) (8 mg) was added. After stirring for 1 hour,the reaction mixture was poured into monosodium citrate (250 mM, 15 mL),extracted with CH₂Cl₂ (10 mL) three times, and the combined organiclayers dried (Na₂SO4) and concentrated to provide the title compound (5mg) as a blue solid: MS expected 809 (C₄₀H₃₆Cl₃N₃O₂S⁺, M+), found 809.

Example 25 Bis(piperidineazepino)-3,5-bissulforhodamine (PBI 3904)

A mixture of11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline (50 mg)and 4-formylbenzene-1,3-disulfonic acid disodium salt hydrate (38 mg) in1 mL of concentrated sulfuric acid was heated with stirring in an openvessel to 100° C. by means of an oil bath for five hours. After thistime, the reaction was removed from the oil bath, and ice was slowlyadded while stirring until liquefied. The acidified water was thendecanted from an oily residue which was further washed 2 more times withwater. The residue was then dissolved in methanol, and the solventevaporated depositing the residue onto celite. The crude product waspurified by silica gel chromatography (gradient of MeOH in CH₂Cl₂) toprovide the title compound as a red solid (78 mg): MS expected 635(C₃₃H₃₅N₂O₇S₂, M+), found 635; λmaxAbs=590 nm (MeOH), λmaxEm=613 nm(MeOH).

Example 26 Bis(piperidineazepino)-5-sulfonylchloride sulforhodamine

A solution of bis(piperidineazepino)-3,5-bissulforhodamine (PBI 3904, 70mg) in POCl₃ (2 mL) and THF (2 mL) was stirred for 1 hour and thenconcentrated under reduced pressure. After stirring another 30 minutes,the reaction mixture was purified by preparative HPLC (gradient of ACNin 0.1% TFA in H₂O) to provide the title compound (1 mg) as a bluesolid: MS expected 769 (C₄₅H₅₅ClN₃O₆ ⁺, M+), found 769.

Example 27Bis(piperidineazepino)-5-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)sulfonyl)sulforhodamine(PBI 3909)

A solution of bis(piperidineazepino)-3,5-bissulforhodamine (PBI 3904, 70mg) in POCl₃ (2 mL) and THF (2 mL) was stirred for 1 hour and thenconcentrated under reduced pressure. This crude sulfonyl chloride wasdissolved in CH₂Cl₂ (5 mL), and triethylamine (0.23 mL) and2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine HCl (43 mg) were added. Afterstirring for 3 days, the reaction mixture was concentrated. The crudeproduct was dissolved in DMF and purified by preparative HPLC (gradientof ACN in 0.1% TFA in H₂O) to provide the title compound (6 mg) as ablue solid: MS expected 840 (C₄₃H₅₄ClN₃O₈S₂ ⁺, M+), found 840.

Example 28 5-methoxy-2-aminonaphthalene

To a solution of 6-aminonaphth-1-ol (1.0 g) in DMF (50 mL), NaH (60% inmineral oil, 0.17 g) was added, and the reaction was stirred for 1 hour.Iodomethane (0.39 mL) was added, and the reaction was stirred foranother 1 hour. The reaction was then partitioned between NaHCO₃ (aq.,150 mL) and EtOAc (100 mL), the layers separated, and the organic layerwashed with brine, dried (Na₂SO₄) and concentrated. The crude reactionwas purified over silica gel (gradient of EtOAc in heptane) to providethe title compound (0.8 g) as an orange oil:: 1H NMR (DMSO-d6) δ 7.81(d, 1H); 7.16 (dd, 1H), 7.04 (d, 1H), 6.85 (dd, 1H), 6.75 (d, 1H), 6.53(dd, 1H), 5.32 (s, 2H), 3.86 (s, 3H).

Example 29 5-hydroxy-2-(dimethylamino)naphthalene

From the purification of 5-methoxy-2-aminonaphthalene5-methoxy-2-(dimethylamino)naphthalene was also isolated. This compoundwas demethylated in the same manner as8-hydroxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine to give the titlecompound: 1H NMR (DMSO-d6) δ 9.72 (s, 1H), 7.93 (d, 1H); 7.10-7.05 (m,3H), 6.82 (d, 1H), 6.52 (dd, 1H), 2.96 (s, 6H); MS expected 188(C₁₂H₁₃NO, M+1), found 188.

Example 309-hydroxy-1,2,3,5,6,7-hexahydrobenzo[f]pyrido[3,2,1-u]quinolone

The title compound was synthesized in a similar manner as11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone startingfrom 5-methoxy-2-aminonaphthalene: 1H NMR (DMSO-d6) δ 9.55 (s, 1H), 7.55(s, 1H); 7.08-7.00 (m, 2H), 6.48 (dd, 1H), 3.14 (q, 4H), 2.86 (q, 2H),2.02-1.86 (m, 4H); MS expected 240 (C₁₆H₁₈NO, M+1), found 240.

Example 31 3-methoxy-1-(dimethylamino)naphthalene

To a solution of 3-methoxy-1-aminonaphthalene (US Patent No. 7,018,431B2, 0.13 g) in DMF (5 mL), K₂CO₃ (0.31 g) and iodomethane (0.09 mL) wereadded. After stirring for 24 hours, the reaction was partitioned betweenwater (30 mL) and EtOAc (30 mL), the layers separated, and the organiclayer washed with brine, dried (Na₂SO₄) and concentrated. The crudereaction purified over silica gel (gradient of EtOAc in heptane) toprovide the title compound (0.14 g) as a clear oil: 1H NMR (DMSO-d6) δ7.99 (d, 1H); 7.74 (d, 1H), 7.40 (ddd, 1H), 7.30 (ddd, 1H), 6.95 (d,1H), 6.67 (d, 1H), 3.83 (s, 3H), 2.78 (s, 6H); MS expected 202(C₁₃H₁₆NO, M+1), found 202.

Example 32 3-hydroxy-1-(dimethylamino)naphthalene

The title compound was synthesized in a similar manner as11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone startingfrom 3-methoxy-1-(dimethylamino)naphthalene: 1H NMR (DMSO-d6) δ 9.53 (s,1H), 7.95 (d, 1H); 7.60 (d, 1H), 7.32 (ddd, 1H), 7.21 (ddd, 1H), 6.75(d, 1H), 6.64 (d, 1H), 2.77 (s, 6H).

Example 33Bis(piperidineazepino)-6-((2-(2-(2-(2-(6-carboxyamido-2-cyanobenzothiazolyl)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)rhodamine(PBI 5122)

To a solution of t-boc-N-amido-dPEG®4-acid (Quanta BioDesign, 1 g) andN-methylmorpholine (0.3 mL) in THF (25 mL), isobutyl chloroformate (0.36mL) was added. After stirring for 30 min, 6-amino-2-cyanobenzothiazole(White et. al., J. Am. Chem. Soc. 88, 2015 (1966), 0.48 g) was added,and the reaction stirred overnight. The reaction was then filtered, andthe eluent concentrated. The crude reaction purified over silica gel(gradient of MeOH in CH₂Cl₂) to provide a clear oil (1.4 g).

The oil from the previous step was dissolved in 15% thioanisole intrifluoroacetic acid (25 mL) at 0° C. The reaction was stirred for 3hours, diluted with diethyl ether and then concentrated to dryness. Thereaction was then filtered, and the eluent concentrated. The crudereaction purified over silica gel (gradient of MeOH in CH₂Cl₂) andimmediately carried on to the next step.

To a solution of bis(piperidineazepino)-6-carboxyrhodamine (76 mg) anddiisopropylethylamine (0.04 mL) in CH2Cl2 (1 mL) was added TSTU (8 mg).After stirring for 15 min, the crude amine from the previous step (62mg) was added. After stirring another 30 min, the reaction mixture waspurified by preparative HPLC (gradient of ACN in 0.1% TFA in H₂O) toprovide the title compound (6 mg) as a blue solid: MS expected 967(C54H59N6O9S+, M+1), found 967.

Example 34 General Procedures for Labeling Oligonucleotides with theDyes of the Present Invention.

A. Oligonucleotide labeling with N-Hydroxysuccinimidyl Ester Dyes

i. 1 μmole Scale

A 5′-amino labeled oligonucleotide was synthesized on an ABI 394 DNAsynthesizer (1 μmole) using 5′ Amino modifier C6 TFA amidite from GlenResearch. /Deprotection was performed in concentrated ammonium hydroxideovernight at 60° C to yield a 5′-aminohexyl labeled oligonucleotide. Theresulting oligonucleotide was evaporated to dryness, redissolved in 1 ml0.5 M NaCl (performed for counter-ion exchange) and desalted on NAP-10size exclusion cartridge (GE Healthcare). After desalting, theoligonucleotide was evaporated to dryness followed by re-dissolution in200 μl 0.5 M sodium carbonate buffer, pH 9.0. The succinimidyl esterdyes (PBI 4451, 4510, 4574, 4563, 4566 and 4509) were dissolved in DMFat a concentration of 20 μl/mg. 2×20 μl aliquots of the dye/DMF solutionwere added to the dissolved oligonucleotide at 30 minutes apart. Afterthe second addition of the dye/DMF solution, the reaction was mixed for1 hour at 20° C. After one hour, it was diluted to 1 ml with water anddesalted on a NAP-10 column (GE Healthcare). The NAP-10 eluate waspurified by reversed phase HPLC on a Phenomonex Jupiter C18 column usingan acetonitrile/0.1M TEAA buffer system. The HPLC purifiedoligonucleotide was evaporated to dryness redissolved in 0.01Mtriethylammonium bicarbonate and desalted on a NAP-10 column. Afterfinal desalt step, the oligonucleotide was evaporated to dryness andstored at −20° C.

ii. 100 μmole Scale

A 5′-amino labeled oligonucleotide was synthesized on an AKTA OligoPilot(100 μmole) DNA synthesizer using 5′ Amino modifier C6 TFA amidite fromGlen Research. Deprotection was performed in concentrated ammoniumhydroxide overnight at 60° C. to yield a 5′-aminohexyl labeledoligonucleotide. The resulting oligonucleotide was evaporated todryness, redissolved in 75 ml 2 M NaCl and desalted on a 500 ml G-25column (GE Healthcare). After desalting, the oligonucleotide wasevaporated to dryness followed by re-dissolution in 50 ml 0.5 M sodiumcarbonate buffer, pH 9.0. The succinimidyl ester dyes (PBI 4451, 4510,4574, 4563, 4566 and 4509) were dissolved in DMF at a concentration of20 μl/mg. 2400 μl of the dye/DMF solution was added drop wise to thedissolved oligonucleotide. The reaction was mixed for 1 hour at roomtemperature. The dye conjugated oligonucleotide was neutralized withsodium acetate, pH 5.5, solution and precipitated from 2× volume ofethanol. The precipitated oligonucleotide was centrifuged at 9000 rpmfor 60 minutes, the supernatant decanted to waste, and the resultingsolid dissolved in 70 ml water and purified by ion-exchangechromatography. The oligonucleotide was concentrated and desalted usingtangential flow ultrafiltration and subsequently evaporated to dryness.It was stored at −20° C.

B. Oligonucleotide Labeling with Phosphoramidites Dyes

i. 1 μmole Scale

A 5′-labeled oligonucleotide was synthesized on an ABI 394 DNAsynthesizer (1 μmole) using the phosphoramidite dye (PBI 3885) of thepresent invention dissolved to 0.1M in acetonitrile. Deprotection wasperformed in t-butylamine/MeOH/water (25/25/50) overnight at 60° C. toyield a 5′-labeled oligonucleotide. The resulting oligonucleotide wasevaporated to dryness, redissolved in 0.01 M triethylammoniumbicarbonate and purified by reversed phase HPLC on a Phenomonex JupiterC18 column using an acetonitrile/0.1 M TEAA buffer system. The HPLCpurified oligonucleotide was evaporated to dryness, redissolved in 0.01M triethylammonium bicarbonate and desalted on a NAP-10 column (GEHealthcare). After final desalt step, the oligonucleotide was evaporatedto dryness and stored at −20 C.

ii. 100 μmole Scale

A 5′-labeled oligonucleotide was synthesized on an AKTA OligoPilot DNAsynthesizer (100 μmole) using the phosphoramidite dye (PBI 3885) of thepresent invention dissolved to 0.1 M in acetonitrile. Deprotection wasperformed in t-butylamine/MeOH/water (25/25/50) overnight at 60° C toyield a 5′-labeled oligonucleotide. The resulting oligonucleotide wasevaporated to dryness, redissolved in 0.01M triethylammonium bicarbonateand purified by ion-exchange HPLC. The resulting purifiedoligonucleotide was concentrated and desalted using tangential flowultrafiltration and evaporated to dryness. The labeled oligonucleotidewas stored at −20° C.

Example 35 PCR and Multiplex PCR using oligonucleotides Labeled with theDyes of the Present Invention

To demonstrate the ability to perform a 6-dye multiplex PCR with thedyes of the present invention, multiplex reactions were performedcontaining primer pairs for 24 STR loci.

For the multiplex reactions, a 5× primer pair master mix for 21 STR loci(“5×21-STR Primer Mix”) as outlined in Table 1 and a 5× reaction mastermix (“5× Reaction Master Mix”) containing reaction buffer and GoTaq® HotStart DNA polymerase were made. Also, in addition to the 21 STR lociprimer pairs, additional primer pairs were prepared as described inTable 2 using dyes of the present invention. These additional primerpairs were made to 150 μM in 1 mM MOPS with 0.1 mM EDTA with final ˜pH7.5 at 25° C.

TABLE 1 Primer Pair Concentration Locus Dye (uM in 5x) Amelogenin 5FAM2.88 D3S1358 5FAM 0.88 D1S1656 5FAM 1.42 D6S1043 5FAM 1.32 D13S317 5FAM1.6 Penta E 5FAM 7.88 Penta D JOE 3.68 D16S539 JOE 2.4 D18S51 JOE 1.18D2S1338 JOE 2.04 CSF1PO JOE 1.4 TH01 ET TMR 1.38 vWA ET TMR 1.8 D21S11ET TMR 1.55 D7S820 ET TMR 2.4 D5S818 ET TMR 2.02 TPOX ET TMR 1.89D8S1179 ET ROX 1.7 D12S391 ET ROX 3.75 D19S433 ET ROX 1.05 FGA ET ROX1.28

TABLE 2 Locus Dye D22S1045 6FAM ET PBI 4510 (624 nm) D2S441 6FAM ET PBI4510 (624 nm) DYS391 6FAM ET PBI 4510 (624 nm) D22S1045 6FAM ET PBI 4563(640 nm) D2S441 6FAM ET PBI 4563 (640 nm) DYS391 6FAM ET PBI 4563 (640nm) D22S1045 6FAM ET PBI 4574 (634 nm) D2S441 6FAM ET PBI 4574 (634 nm)DYS391 6FAM ET PBI 4574 (634 nm)

Multiplex reactions were then set up as follows:

-   -   A. Multiplex 1 Mix (for 10 reactions):    -   5× 21-STR Primer Mix: 50 μl    -   5×Reaction Master Mix: 50 μl    -   4510-D22S1045 primer pair (0.6 μM): 1μl    -   4510-D2S441 primer pair (0.6 μM): 1 μl    -   4510-DYS391 primer pair (0.6 μM): 1 μl    -   Nuclease free water: 137 μl    -   B. Multiplex 2 Mix (for 10 reactions):    -   5× 21-STR Primer Mix: 50 μl    -   5× Reaction Master Mix: 50 μl    -   4563-D22S1045 primer pair (0.6 μM): 1 μl    -   4563-D2S441 primer pair (0.6 μM): 1 μl    -   4563-DYS391 primer pair (0.6 μM): 1 μl    -   Nuclease free water: 137 μl    -   C. Multiplex 3 Mix (for 10 reactions):    -   5× 21-STR Primer Mix: 50 μl    -   5× Reaction Master Mix: 50 μl    -   4574-D22S1045 primer pair (0.6 μM): 1 μl    -   4574-D2S441 primer pair (0.6 μM): 1 μl    -   4574-DYS391 primer pair (0.6 μM): 1 μl    -   Nuclease free water: 137 μl    -   D. Multiplex 4 Mix (for 10 reactions):    -   5× 21-STR Primer Mix: 50 μl    -   5× Reaction Master Mix: 50 μl    -   4510-D22S1045 primer pair (2.4 μM): 4 μl    -   4510-D2S441 primer pair (2.4 μM): 4 μl    -   4510-DYS391 primer pair (2.4 μM): 4 μl    -   Nuclease free water: 128 μl    -   E. Multiplex 5 Mix (for 10 reactions):    -   5× 21-STR Primer Mix: 50 μl    -   5× Reaction Master Mix: 50 μl    -   4563-D22S1045 primer pair (2.4 μM): 4 μl    -   4563-D2S441 primer pair (2.4 μM): 4 μl    -   4563-DYS391 primer pair (2.4 μM): 4 μl    -   Nuclease free water: 128 μl    -   F. Multiplex 3 Mix (for 10 reactions):    -   5× 21-STR Primer Mix: 50 μl    -   5× Reaction Master Mix: 50 μl    -   4574-D22S1045 primer pair (2.4 μM): 4 μl    -   4574-D2S441 primer pair (2.4 μM): 4 μl    -   4574-DYS391 primer pair (2.4 μM): 4 μl    -   Nuclease free water: 128 μl

24 μl of each multiplex mix was then added to a well of a 96-well PCRplate. 1 μl of 1 ng/μL male DNA (2800M Promega Cat. # DD7101 or 9948Promega Cat. # DD206A) or 0.5 ng/μL male DNA (C274 or QC2; Promega) wasadded to each well. Various single-source male DNA samples were used todetermine variability in balance and bleedthrough/bridging of the dyeswith various allele patterns. Reactions were then run on an AppliedBiosystems 9700 thermal cycler using the following cycling conditions:96° C. for 1 minutes; then 30 cycles of 94° C. for 10 seconds, 59° C.for 1 minutes and 72° C. for 30 seconds; 60° C. for 10 minutes; and a 4°C. soak. Reactions were then analyzed on an Applied Biosystems 3500xLGenetic Analyzer (FIGS. 4-8).

This example demonstrates that the dyes of the present invention workvery well with respectable signals and very little bleedthrough orbridging in multiplex PCR.

Example 36 Cell Labeling using the Dyes of the Present Invention

To determine the ability of the dyes of the present invention to be usedfor labeling in cells, the dyes were conjugated to a HaloTag® ligand(Promega) to monitor activity/movement of the HaloTag® protein orHaloTag® fusion protein. For cell labeling, the ligands #3780, 3781,3782, 3783, 3905, 3906, 3954, 4356 and 4357 (Table 3) were used. U2OScells stably expressing HaloTag® protein in the nucleus (HT-NLS) wereused to test for the cell permeability of the ligands. In some cases,the efficiency of the removal of unbound ligand was also determinedLigands for which HT-NLS imaging did not show obvious ligand removalissues were further tested in cells stably expressing HaloTag® proteinin their cytoplasm using a p65-HaloTag® fusion (p65-HT) and U2OS cellsnot expressing HaloTag® protein. The U2OS p65-HT stable cells were usedto establish the imaging parameters for a medium to low expressingfusion protein. These same parameters were then used to assess removalof unbound ligand in U2OS cells not expressing HaloTag. All ligands werediluted in DMSO to 10 mM prior to use with cells.

TABLE 3 Ligand Number Ligand Structure Dye Number 3780

3781

PBI 3739 3782

PBI 3762 3783

PBI 3769 3905

PBI 3762 3906

PBI 3763 3954

PBI 3954 4356

4357

For all imaging experiments, U2OS cells were plated in Lab-Tek II CG(Nunc) chambered coverslips and left overnight at 37° C.+5% CO₂ toattach. Cells were exposed to 1 μM ligand by a rapid label protocol.Briefly, cells were exposed to the ligand for 15 minutes in the presenceof ATCC-recommended complete media at 37° C.+5% CO₂ and 800 μg/ml G418(Promega). After labeling, cells were rinsed 3 times with complete mediaand incubated for 30 minutes at 37° C.+5% CO₂. The media was thenreplaced with fresh complete media, and cells transferred to a confocalmicroscope for imaging.

In some cases, U2OS cells stably expressing HT-NLS were labeled by ano-wash protocol. Briefly, cells were exposed to 100 nM ligand overnightat 37° C.+5% CO₂. In these cases, the ligand was added at the time ofcell plating. On the following day, the ligand containing media wasreplaced with fresh complete media, and cells were transferred to aconfocal microscope for imaging.

Confocal images were acquired using an Olympus Fluoview FV500 confocalmicroscope (Olympus, USA) outfitted with a 37° C.+CO₂ environmentalchamber (Solent Scientific Ltd., UK) and appropriate filter sets. SeeFIGS. 9-16.

In order to quantify labeling efficiency of the ligands, SDS-PAGEanalysis was performed. Briefly, cells were plated as above for imagingand were first labeled with 1 μM of ligand for 15 minutes at 37° C.+5%CO₂. The ligand-containing media was then replaced with media containing5 μM HaloTag® TMR ligand (Promega; Cat. No. G8252) and incubated for 15minutes at 37° C.+5% CO₂. Cells were then rinsed 3 times and washed for30 minutes at 37° C.+5% CO₂. The cells were then rinsed once with 1×PBS,collected in 1× SDS gel loading buffer (4× buffer (0.24M Tris, 2% SDS,50.4% Glycerol, 0.4M DTT, 3 mM Bromophenol Blue and Hydrochloric Acid topH6.8) diluted in water), placed at 95° C. for 5 minutes and loaded on a4-20% Tris-Glycine precast gel (Invitrogen). The gel was then scannedusing a Typhoon 9410 (Amersham Biosciences) (FIG. 17).

To determine cell viability after labeling, cells were plated in whitetissue culture treated Costar 96-well plates (Fisher Scientific). Boththe CellTiter-Glo® Luminescent Cell Viability Assay and HaloTag® proteinarrays were performed as per manufacturer protocols (Promega). Briefly,for the CellTiter-Glo assay, 100 μl of the CellTiter-Glo reagent wasadded to the 100 μl of media containing cells. The contents were mixedon an orbital shaker for 2 minutes and incubated at room temperature for10 minutes. Luminescence was measured using a GloMax® Multi DetectionSystem (Promega). The luminescent signal generated is directlyproportional to the amount of ATP present in the sample which isdirectly proportional to the number of cells present in the culture.

In order to assess the use of ligand 3782 for gel based analysis, itsperformance was compared to that of the HaloTag® TMR Ligand (PromegaCat. No. G8252). To do this, the standard Promega protocol for labelingproteins expressed in a cell-free system was used. Briefly, each ligandwas diluted to 10 μM in 1× PBS, and 1μl of each ligand added to 2 μleach of HaloTag®-GST Standard Protein (Promega) or HaloTag®-protein Gpurified from E.coli. 7 μl of 1×PBS was then added to each labelingreaction for a total volume of 10 μl, and reactions were incubated for30 minutes at room temperature protected from light. 5 μl of eachlabeling reaction was then added to 5 μl of 2× SDS gel loading bufferand heated to 70° C. for 2 minutes. The samples were then loaded and runon a SDS-polyacrylamide gel and visualized using a fluorescent scanneras described above (FIGS. 18 and 19).

Example 37 Synthesis of5-((2-(4-(3-tert-Butyl-5-(3-phenylureido)-1H-pyrazol-1-yl)benzylamino)-2-oxoethyl)carbamoyl)-2-bis(piperidineazepino)rhodamine(PBI-4838)

The title compound was synthesized usingbis(piperidineazepino)-6-carboxyrhodamine and1-(1-(4-((2-aminoacetamido)methyl)phenyl)-3-tert-butyl-1H-pyrazol-5-yl)-3-phenylurea(Tecle et al. 2009, Chem. Biol. Drug Des., 74:547-559) in a mannersimilar to PBI-3781 (Example 21): MS expected 815 (C₅₈H₆₁N₈O₆ ⁺, M+),found 815.

Example 38 Bioluminescence Resonance Energy Transfer (BRET) using PBI4828

To demonstrate the ability of the dyes of the present invention to beused in BRET applications, a fluorescent dye tracer comprising a dye ofthe present invention, PBI 4838, was generated to monitor binding of aknown drug to a kinase target in living cells. In this example, ap38alpha kinase inhibitor, BIRB796, was used as a scaffold to generate afluorescent tracer for inactive p38alpha kinase comprising PBI 4838(Tecle et al. 2009. Chem Biol Drug Des: 74: 547-559; FIG. 20). Thetracer was then applied to living or lysed cells expressing a NanoLucluciferase-p38alpha kinase fusion protein. Upon addition of a furimazinesubstrate for the NanoLuc luciferase, dose-dependent BRET was thenmeasured in both living and lysed cells.

HEK293 cells (20,000 cells per well in 96-well format) were transientlytransfected (Fugene HD, Promega Corporation) with pF5 plasmid DNA(Promega Corporation) encoding a NanoLuc® luciferase-p38alpha kinasefusion protein. As a negative control, some cells were transfected witha pF5 plasmids DNA encoding a NanoLuc luciferase-PKC alpha fusionprotein. On the second day post transfection, the cell medium wasreplaced with serum-free Opti-MEM (Life Technologies) with or without 50ug/ml digitonin.

For tracer saturation experiments, cells were treated with seriallydiluted PBI 4838 in the presence or absence of a molar excess of BIRB796(10uM final).

For BIRB796 competition experiments, serially-diluted BIRB796 wasapplied to cells in the presence or absence of 0.5uM PBI 4838 (finalconcentration).

Cells were then allowed to equilibrate with tracer and BIRB796 for 2hours at 37° C. A furimazine substrate (PBI 3939), a substrate for theNanoLuc luciferase, then was added to cells to a final concentration of20uM. BRET ratios were recorded using a Varioskan luminometer at thefollowing wavelengths: 630 nm (acceptor)/450 nm (donor). Acceptor/donorvalues were used to determine BRET ratio (FIGS. 21 and 22).

The data demonstrates that the dyes of the present invention can be usedin BRET applications. In the tracer saturation experiments, live orpermeablized cells expressing NanoLuc-p38alpha were incubated withserially diluted PBI 4838 resulting in dose-dependent increase in BRET.The results indicate binding of PBI 4838 to NanoLuc fusion proteins inliving cells. In the presence of a vast molar excess of unlabeledBIRB796, the BRET signal was inhibited completely, indicating thatnearly the entire BRET signal between NanoLuc-p38alpha and PBI 4838 isspecific. For the BIRB796 competition experiments, the data shows theability to competitively displace a fixed concentration of PBI-4838 in adose-dependent manner with unlabeled BIRB796. The control experimentsfurther support the specificity of the specific BRET signal between PBI4838 and NanoLuc p38alpha. This demonstrates the use of cells expressingNanoLuc fused to an irrelevant kinase. In the control experiments, cellsexpressing NanoLuc-PKCalpha show only a trace amount of BRET signal inthe presence of PBI 4838, which is not affected by unlabeled BIRB796.

Example 39 Bioluminescence Resonance Energy Transfer (BRET) using PBI3781

To demonstrate the ability of the dyes of the present invention to beused in BRET applications, a fluorescent dye of the present inventionwas conjugated to a chloroalkane as in Example 21. The dye-chloroalkaneconjugate PBI 3781 covalently binds to a HaloTag® protein or a HaloTag®fusion protein allowing detection and/or measurement of the HaloTag orHaloTag® fusion protein. PBI 3781 was used in combination with HaloTag®and NanoLuc fusion proteins to measure protein-protein interactions inliving cells via BRET.

In this example, the rapamycin-mediated interaction between a NanoLucluciferase-FK506 binding domain of mTOR (Frb) fusion and aHaloTag-FKBP12 fusion (FK506 binding protein) was used to demonstrate aninducible protein-protein interaction. The interaction was measured as arapamycin-dependent increase of BRET occurring between the NanoLuc-FRBfusion (donor) and HaloTag-FKBP12 fusion bound to PBI 3781 (acceptor)(FIG. 23A).

HeLa cells were co-transfected with pF5-based constructs (PromegaCorporation) for the expression of Frb-NanoLuc or FRKBP12-HaloTag®fusion proteins using FuGENE® HD Transfection Reagent according to themanufacturer's instructions (Promega Corporation). The cells were thenincubated overnight at 37° C., 5% CO₂.

One day following transfection, the cells were collected and re-platedinto wells of a white, 96-well tissue culture plate at 200,000 cells/well in 100 μL DMEM+10% FBS and incubated for 24 hours at 37° C., 5%CO₂.

Two days after transfection, the growth medium was replaced with phenolred-free DMEM +5% FBS containing 500 nM of PBI 3781, and the cellsincubated for 120 minutes 37° C., 5% CO₂. The cells were then treatedwith 50 μL of a serial dilution of rapamycin in phenol-red-free DMEM andincubated for 15 minutes at room temperature. 50 μL, 40 mM furimazine inphenol-red-free DMEM was added, and BRET measured using a ThermoVarioskan plate reader (500 msec integration time; donor channelemission 450/60 bandpass filter; acceptor channel emission 610 nmlongpass color glass filter) (FIG. 24).

This experiment demonstrates a concentration-dependent change ofabsolute BRET signal following the addition of a serial dilution ofrapamycin. The experiment also demonstrates that a dye of the presentinvention can be used to detect other intracellular protein-proteininteractions using suitable pairs of NanoLuc and HaloTag® fusionproteins in combination with dye-chloroalkane HaloTag® ligand, e.g., PBI3781.

Example 40 Prophetic Example of Using a Dye-Cyanobenzothiazole Conjugate

The interaction of a ligand, such as epidermal growth factor (EGF) toepidermal growth factor receptor (EGFR), can be measured in whole cellpopulations. In such an example, a HaloTag-EGF fusion protein, whichcontains a TEV protease cleavage site between the HaloTag and EGFprotein domains in which the last residue of TEV protease cleavage siteencodes a cysteine residue, can be used. Upon cleavage with TEVprotease, a Cys-EGF protein is generated and may be reacted with a CBTlabeled compound such as PBI 5122 (Example 33). The PBI 5122-Cys-EGF canserve as a probe capable of binding to a NanoLuc-EGFR fusion proteinexpressed in living cells. Upon binding in close proximity, energytransfer (BRET) can occur, leading to increased dye emission. In anotherconfiguration, unlabeled EGF, or ligands of similar binding mechanism,may disrupt the PBI 5122-EGF: NanoLuc-EGFR complex, leading to adecrease in energy transfer. The compatibility of these labelingchemistries and energy transfer methods could allow for thequantification of ligand binding events from ligands and receptorsgenerated in whole cells.

1. A method to detect an interaction between a biomolecule and a proteinof interest comprising: a) contacting a sample suspected of containingthe protein of interest with a composition comprising a conjugatecomprising a compound and the biomolecule; and b) detecting the presenceor amount of the compound, thereby detecting the interaction between theprotein of interest and the biomolecule; wherein the compound is acompound according to formula (IIIa), (IIIb) or (IIIc):

wherein R¹¹ is independently H or C₁₋₄ alkyl, L-R or L-C_(s); L is acovalent linkage that is linear or branched, cyclic or heterocyclicsaturated or unsaturated, having 1-16 non hydrogen atoms such that thelinkage contains any combination of ester, acid, amine, amide, alcohol,ether, thioether or halide groups or single, double, triple or aromaticcarbon-carbon bond; R is a reactive group; C_(s) is a conjugatedsubstance selected from the group consisting of solid supports, resinparticles, beads, assay plates, proteins, nucleotides, polynucleotides,enzyme substrates, antibodies, nanobodies, polypeptides,polypeptide-based toxins, amino acids, lipids, carbohydrates, haptens,drugs, ion-complexing agents, microparticles, polymers, cells, viruses,fluorophores, chloroalkanes, and cyanobenzothiazoles; R² and R¹⁶ can beindependently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H,L-R or L-C_(s); R³ and R⁴ are H, alkyl, L-R, L-C_(s), L-CO₂H, L-SO₃H ortogether form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;alternatively, R² and R³ and independently R⁴ and R¹⁶ together form acarbocyclic, heterocyclic, aryl or heteroaryl ring; R⁵, R¹², R¹³, R¹⁴and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s); R²⁰, R²¹, R²² and R²³ are independentlyH or C₁₋₆ alkyl or one or more of R²⁰ and R²¹, R²¹ and R²², R²² and R²³,together form an aryl, heteroaryl, carbocyclic or heterocyclic ring; R¹¹and R¹² may together form a carbocyclic, heterocyclic, aryl orheteroaryl ring; R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl,aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s); X isCHR²³, O, S or NR³⁰; and R³⁰ is H, C₁₋₄ alkyl or —C(O)C₁₋₄ alkyl.
 2. Themethod of claim 1, wherein the protein of interest is fused to areporter protein.
 3. The method of claim 2, wherein the reporter proteinis a luciferase protein.
 4. The method of claim 1, wherein the labeledbiomolecule is a tracer.
 5. The method of claim 1, wherein theinteraction is detected by bioluminescence reasonance energy transfer.6. The method of claim 1, wherein the protein of interest is expressedin a cell.
 7. The method of claim 1, wherein at least one of R2-16 is-L-CS.
 8. A method to detect a selected molecule in a sample,comprising: a) contacting a sample suspected of containing the selectedmolecule with a composition comprising a conjugate comprising a compoundand a ligand for the selected molecule so as to yield a mixture; and b)detecting the presence or amount of the compound in the mixture, therebydetecting the selected molecule in the sample; wherein the compound is acompound according to formula (IIIa), (IIIb) or (IIIc):

wherein R¹¹ is independently H or C₁₋₄ alkyl, L-R or L-C_(s); L is acovalent linkage that is linear or branched, cyclic or heterocyclicsaturated or unsaturated, having 1-16 non hydrogen atoms such that thelinkage contains any combination of ester, acid, amine, amide, alcohol,ether, thioether or halide groups or single, double, triple or aromaticcarbon-carbon bond; R is a reactive group; C_(s) is a conjugatedsubstance selected from the group consisting of solid supports, resinparticles, beads, assay plates, proteins, nucleotides, polynucleotides,enzyme substrates, antibodies, nanobodies, polypeptides,polypeptide-based toxins, amino acids, lipids, carbohydrates, haptens,drugs, ion-complexing agents, microparticles, polymers, cells, viruses,fluorophores, chloroalkanes, and cyanobenzothiazoles; R² and R¹⁶ can beindependently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H,L-R or L-C_(s); R³ and R⁴ are H, alkyl, L-R, L-C_(s), L-CO₂H, L-SO₃H ortogether form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;alternatively, R² and R³ and independently R⁴ and R¹⁶ together form acarbocyclic, heterocyclic, aryl or heteroaryl ring; R⁵, R¹², R¹³, R¹⁴and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s); R²⁰, R²¹, R²² and R²³ are independentlyH or C₁₋₆ alkyl or one or more of R²⁰ and R²¹, R²¹ and R²², R²² and R²³,together form an aryl, heteroaryl, carbocyclic or heterocyclic ring; R¹¹and R¹² may together form a carbocyclic, heterocyclic, aryl orheteroaryl ring; R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl,aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s); X isCHR²³, O, S or NR³⁰; and R³⁰ is H, C₁₋₄ alkyl or —C(O)C₁₄ alkyl.
 9. Themethod of claim 8, wherein at least one of R²⁻¹⁶ is -L-C_(s).
 10. Themethod of claim 8, wherein the ligand is an oligonucleotide.
 11. Amethod of detecting the presence of a nucleic acid polymer in a samplecomprising: a) contacting a sample suspected of containing a nucleicacid polymer with a composition comprising a conjugate comprising acompound and an oligonucleotide; and b) detecting the presence or amountof the compound in the sample, thereby detecting the presence of thenucleic acid polymer in the sample: wherein the compound is a compoundaccording to formula (IIIa), (IIIb) or (IIIc):

wherein R¹¹ is independently H or C₁₄ alkyl, L-R or L-C_(s); L is acovalent linkage that is linear or branched, cyclic or heterocyclicsaturated or unsaturated, having 1-16 non hydrogen atoms such that thelinkage contains any combination of ester, acid, amine, amide, alcohol,ether, thioether or halide groups or single, double, triple or aromaticcarbon-carbon bond; R is a reactive group; C_(s) is a conjugatedsubstance selected from the group consisting of solid supports, resinparticles, beads, assay plates, proteins, nucleotides, polynucleotides,enzyme substrates, antibodies, nanobodies, polypeptides,polypeptide-based toxins, amino acids, lipids, carbohydrates, haptens,drugs, ion-complexing agents, microparticles, polymers, cells, viruses,fluorophores, chloroalkanes, and cyanobenzothiazoles; R² and R¹⁶ can beindependently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H,L-R or L-C_(s); R³ and R⁴ are H, alkyl, L-R, L-C_(s), L-CO₂H, L-SO₃H ortogether form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;alternatively, R² and R³ and independently R⁴ and R¹⁶ together form acarbocyclic, heterocyclic, aryl or heteroaryl ring; R⁵, R¹², R¹³, R¹⁴and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s); R²⁰, R²¹, R²² and R²³ are independentlyH or C₁₋₆ alkyl or one or more of R²⁰ and R²¹, R²¹ and R²², R²² and R²³,together form an aryl, heteroaryl, carbocyclic or heterocyclic ring; R¹¹and R¹² may together form a carbocyclic, heterocyclic, aryl orheteroaryl ring; R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl,aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s); X isCHR²³, O, S or NR³⁰; and R³⁰ is H, C₁₄ alkyl or —C(O)C₁₄ alkyl.
 12. Themethod of claim 11, wherein more than one nucleic acid polymer in asample is detected in a single reaction.
 13. The method of claim 12,wherein the more than one nucleic acid polymer is detected by capillaryelectrophoresis.
 14. A method of labeling a biomolecule comprising: a)contacting a sample suspected of containing the biomolecule with acomposition comprising a compound so as to yield a mixture; and b)detecting the presence or amount of the compound, thereby detecting thepresence or amount of the labeled biomolecule in the mixture; whereinthe compound is a compound according to formula (IIIa), (IIIb) or(IIIc):

wherein R¹¹ is independently H or C₁₋₄ alkyl, L-R or L-C_(s); L is acovalent linkage that is linear or branched, cyclic or heterocyclicsaturated or unsaturated, having 1-16 non hydrogen atoms such that thelinkage contains any combination of ester, acid, amine, amide, alcohol,ether, thioether or halide groups or single, double, triple or aromaticcarbon-carbon bond; R is a reactive group; C_(s) is a conjugatedsubstance selected from the group consisting of solid supports, resinparticles, beads, assay plates, proteins, nucleotides, polynucleotides,enzyme substrates, antibodies, nanobodies, polypeptides,polypeptide-based toxins, amino acids, lipids, carbohydrates, haptens,drugs, ion-complexing agents, microparticles, polymers, cells, viruses,fluorophores, chloroalkanes, and cyanobenzothiazoles; R² and R¹⁶ can beindependently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H,L-R or L-C_(s); R³ and R⁴ are H, alkyl, L-R, L-C_(s), L-CO₂H, L-SO₃H ortogether form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;alternatively, R² and R³ and independently R⁴ and R¹⁶ together form acarbocyclic, heterocyclic, aryl or heteroaryl ring; R⁵, R¹², R¹³, R¹⁴and R¹⁵ are independently H, alkyl, aryl, heteroaryl, CO₂H, SO₃H,L-CO₂H, L-SO₃H, L-R or L-C_(s); R²⁰, R²¹, R²² and R²³ are independentlyH or C₁₋₆ alkyl or one or more of R^(2°)and R²¹, R²¹ and R²², R²² andR²³, together form an aryl, heteroaryl, carbocyclic or heterocyclicring; R¹¹ and R¹² may together form a carbocyclic, heterocyclic, aryl orheteroaryl ring; R⁶⁻¹⁰ are independently H, F, Cl, Br, I, OH, alkyl,aryl, heteroaryl, CO₂H, SO₃H, L-CO₂H, L-SO₃H, L-R or L-C_(s); X isCHR²³, O, S or NR³⁰; and R³⁰ is H, C₁₋₄ alkyl or —C(O)C₁₄ alkyl.
 15. Themethod of claim 14, wherein the compound contains a cyanobenzothiazole.16. The method of claim 14, wherein the biomolecule is a protein. 17.The method of claim 16, wherein the protein comprises an N-terminalcysteine.
 18. The method of claim 17, wherein the protein comprising anN-terminal cysteine is labeled with the compound comprising acyanobenzothiazole.